2&#39;,5&#39;-phosphorothioate/phosphodiester oligoadenylates and anti-viral uses thereof

ABSTRACT

Optically active antiviral compounds having the formula ##STR1## wherein m is 0, 1, 2, or 3; n and q are selected from the group of 0 and 1, provided that n and q may not both be zero; and R, R 1 , and R 2  are independently of each other selected from the group consisting of oxygen and sulfur, provided that all R, R 1  and R 2 , may not be oxygen, and further provided that all R, R 1 , and R 2  may not be sulfur. The compounds possess increased antiviral activity and/or metabolic stability.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was made, in part, in the course of worksupported by National Science Foundation grant DMB-9004139 and U.S.Public Health Service grant P30-CA12227.

This is a continuation of application Ser. No. 08/306,273 filed on Sep.14. 1994 now abandoned.

FIELD OF THE INVENTION

This invention relates to synthetic analogues of naturally occurringantiviral 2',5'-oligoadenylates wherein at least one of theinternucleotide phosphodiester linkages is replaced with opticallyactive phosphorothioate groups. The compounds with selectedinternucleotide phosphodiester linkages have antiviral activity andincreased metabolic stability.

BACKGROUND OF THE INVENTION

The full nomenclature of the subject matter of the present inventioninvolves lengthy terms. It is customary for those skilled in the art toabbreviate oligoadenylate analogues and related terms in a mannerwell-known to the art. These general and customary abbreviations are setforth herein below and may be utilized in the text of thisspecification.

Abbreviations

2-5A, 2',5'-oligoadenylate or p₃ A_(n) : Oligomer of adenylic acid with2',5'-phosphodiester linkages and a 5'-terminal triphosphate group.

A₂, A₃ and A₄ : Dimer, trimer and tetramer of adenylic acid with2',5'-phosphodiester linkages.

pA₃, ppA₃ (or p₂ A₃), pppA₃ (or p₃ A₃): 5'-terminal mono-, di- andtriphosphates of A₃.

pA₄, ppA₄ (or p₂ A₄), pppA₄ (or p₃ A₄): 5'-terminal mono-, di- andtriphosphates of A₄.

ApA: Dimer of adenylic acid with 2'-5'-phosphodiester linkage.

Ap*A: Dimer of adenylic acid with 2'-5'-phosphorothioate linkage.

PR: The R stereoconfiguration about a chiral phosphorous atom in aphosphorothioate internucleotide linkage.

PS: The S stereoconfiguration about a chiral phosphorous atom in aphosphorothioate internucleotide linkage.

A_(Rp) *ApA: (PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.

A_(Sp) *ApA: (PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.

ApA_(Rp) *A: Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenosine.

ApA_(Sp) *A: Adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine.

pA_(Rp) *ApA, ppA_(Rp) *ApA, pppA_(Rp) *ApA, pA_(Sp) *ApA, ppA_(Sp)*ApA, pppA_(Sp) *ApA, pApA_(Rp) *A, ppApA_(Rp) *A, pppApA_(Rp) *A,pApA_(Sp) *A, ppApA_(Sp) *A, pppApA_(Sp) *A: 5'-mono-, di- andtriphosphates of A_(Rp) *ApA, A_(Sp) *ApA, ApA_(Rp) *A, and ApA_(Sp) *A.

A_(Rp) *ApApA:(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenylyl-(2',5')-adenosine.

A_(Sp) *ApApA:(PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenylyl-(2',5')-adenosine.Id.1 ApA_(Rp) *ApA: Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.

ApA_(Sp) *ApA:Adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.

ApApA_(Rp) *A:Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.

ApApA_(Sp) *A:Adenylyl-(2',5')-adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine.

pA_(Rp) *ApApA, ppA_(Rp) *ApApA, pppA_(Rp) *ApApA, pA_(Sp) *ApApA,ppA_(Sp) *ApApA, pppA_(Sp) *ApApA, pApA_(Rp) *ApA, ppApA_(Rp) *ApA,pppApA_(Rp) *ApA, pApA_(Sp) *ApA, ppApA_(Sp) *ApA, pppApA_(Sp) *ApA,pApApA_(Rp) *A, ppApApA_(Rp) *A, pppApApA_(Rp) *A, pApApA_(Sp) *A,ppApApA_(Sp) *A, pppApApA_(Sp) *A: 5'-mono-, di- and triphosphates ofthe above tetramers.

bz: benzoyl

ce: cyanoethyl

CFS: Chronic Fatigue Syndrome

DEAE: 2-(diethylamino)ethyl

DBU: 1.8 diazabicyclo 5.3.0!undec-7-enc

HIV: Human Immunodeficiency Virus

MeOTr: monomethoxytrityl

M.O.I: multiplicity of infection

mRNA: Messenger RNA

npe: 2-(4-niytophenyl)ethyl

PBL: Peripheral blood lymphocytes

pCp: Cytidine 3'-5'-bisphosphate

(PS)-ATP-alpha-S: Adenosine 5'O--(PS)-(1-thiotriphosphate).

RNase L: 2-5A-dependent endoribonuclease.

rRNA: Ribosomal RNA

RT: Reverse transcriptase

SCP: Specific cleavage products

tbds: (tert-butyl)dimethylsilyl

Tris: tris(hydroxymethyl)aminomethane

tRNA: Transfer RNA

It is generally regarded that activation of RNase L by 2-5A is key tothe antiviral defense mechanisms. Interferon induces transcription ofthe enzyme 2-5A synthetase which produces 2',5' linked oligoadenylatesupon activation of double-stranded RNA. Previously, the only knownbiochemical effect of 2-5A is activation of RNase L. This enzymehydrolyses mRNA and rRNA, thereby resulting in inhibition of proteinsynthesis. The activation of RNase L is transient unless 2-5A iscontinuously synthesized, since 2-5A is rapidly degraded. RNase Lactivation thus plays a critical role in inhibiting replication, andtherefore in defending against infection by viruses.

A correlation has also been established between 2-5A metabolism and thegrowth cycle of HIV-1, i.e., high levels of 2-5A and activated RNase Lcorrelate with failure of infected cells to release HIV-1, Schroder etal., J. Biol. Chem. 264: 5669-5673 (1989). Conversely, when theintracellular 2-5A pool decreases, RNase L can not be activated andHIV-1 production increases. A role for 2-5A cores as inhibitors of HIV-1replication has been established with reports that 2-5A trimer andtetramer cores, 5'-monophosphates and 5'-triphosphates inhibit HIV-1reverse transcriptase/primer complex formation, Montefiori et al., Proc.Natl. Acad. Sci. USA 86: 7191-7194 (1989); Muller et al., Biochemistry30: 2027-2033 (1991); Sobol et al., Biochemistry 32: 1211-12118 (1993).

The introduction of the phosphorothioate group in the2',5'-internucleotide linkages of 2-5A, induces metabolic stabilitygreater than authentic 2-5A and resulted in the first 2-5A cores (i.e.2-5A lacking 5'-phosphate moieties) able to activate RNase L (Kariko etal. Biochemistry 26: 7136-7142 (1987); Charachon et al. Biochemistry 29:2550-2556 (1990)). Further, RNase L is a functionally stereoselectiveenzyme and 2-5A trimers and tetramers having at least one of theinternucleotide phosphorothioate 2',5'-linkages of the PS configurationhave greatly enhanced metabolic stability. The chemical synthesis of thefully resolved 2',5'-phosphorothioate adenylate trimer and tetramercores has been reported, Suhadolnik et al., U.S. Pat. No. 4,924,624.Preparation of the stereoisomers via enzymatic synthesis is not possibledue to the sterospecificity of 2-5A synthetase for the substrate(PS)-ATP-alpha-S, which yields trimer and tetramer products of the PRconfiguration exclusively. Further, while Lebleu et al., U.S. Pat. No.4,981,957, discloses the enzymatic synthesis of aphosphorothioate-substituted derivative of 2',5' oligoadenylate, thecompounds disclosed are not stereospecific.

SUMMARY OF THE INVENTION

Compounds of the present invention useful in inhibiting viral infectionsin plants and mammals have increased metabolic stability and/orantiviral activity.

The compounds and the water-soluble salts thereof are of the formula##STR2## wherein m is zero, 1, 2 or 3; n and q are selected from thegroup of zero and 1, provided that n and q may not both be zero; and R,R₁ and R₂ are independently selected from the group of oxygen andsulfur, provided that all R, R₁ and R₂, may not be oxygen, and furtherprovided that all R, R₁ and R₂ may not be sulfur.

The invention also comprises a method of inhibiting viral infection inmammals or plants by administering an antivirally effective amount of acompound according to the above formula, or a water-soluble saltthereof, and antiviral compositions containing such compounds with acarrier.

Compounds according to the formula wherein n is 1 and q is 1 may beutilized to form oligoadenylate conjugates with the macromolecularcarrier poly(L-lysine) for intracellular transport. Suchpoly(L-lysine)/2',5'-phosphorothioate/phosphodiester oligoadenylateconjugates have the formula ##STR3## wherein q is an integer from about60 to about 70, and R is randomly R' or ##STR4## From about five toabout ten of the R groups comprise R'. R' has the following formulawherein m is 0, 1, 2 or 3; and where each R₃, R₄, or R₅ areindependently selected from the group of oxygen and sulfur; providedthat all R₃, R₄ or R₅ may not be oxygen; and further provided that allR₃, R₄ or R⁵, may not be sulfur. ##STR5##

Preferably, at least one of the internucleotide phosphorothioate groups##STR6## of the poly(L-lysine)/2',5'-phosphorothioate/phosphodiesteroligoadenylate conjugates is of the PR configuration.

DESCRIPTION OF THE FIGURES

FIG. 1A represents the results of a radiobinding assay indicating theability of 2',5'-phosphorothioate/phosphodiester trimer core 2-5Aderivatives to activate partially-purified RNase L from L929 cellextracts to hydrolyze the substrate poly(U) ³² P!pCp, but not poly(C).Activation of RNase L was determined by the conversion of poly(I) ³²P!pCp to acid soluble fragments. 100% represents 25,000 dpm of poly(U)³² P!pCp bound to glass fiber filters. 2-5A oligomer (phosphodiesterinternucleotide linkage) is included for comparison. The curves arelabeled as follows: p₃ A₃ (); A₃ (♦); A_(Rp) *ApA (□); A_(Sp) *ApA (▪);ApA_(Rp) *A (Δ); and ApA_(Sp) *A (▴)

FIG. 1B represents the results of a radiobinding assay performedaccording to the method of FIG. 1A for2',5'-phosphorothioate/phosphodiester trimer 5'-monophosphate 2-5Aderivatives. The curves are labeled as follows: p₃ A₃ (); pA₃ (∘);pA_(Rp) *ApA (□); pA_(Sp) *ApA (▪); pApA_(Rp) *A (Δ); and pApA_(Sp) *A(▴).

FIG. 1C represents the results of a radiobinding assay performedaccording to the method of FIG. 1A for2',5'-phosphorothioate/phosphodiester tetramer core-2-5A derivatives.The curves are labeled as follows: p₃ A₄ (); A_(Rp) *ApApA (∇); A_(Sp)*ApApA (▾); ApA_(Rp) *ApA (□); ApA_(Sp) *ApA (▪); ApApA_(Rp) *A (Δ); andApApA_(Sp) *A (▴).

FIG. 1D represents the results of a radiobinding assay performedaccording to the method of FIG. 1A for2',5'-phosphorothioate/phosphodiester tetramer 5'-monophosphate 2-5Aderivatives. The curves are labeled as follows: p₃ A₄ (); pA₄ (∘);pA_(Rp) *ApApA (∇); pA_(Sp) *ApApA (▾); pApA_(Rp) *ApA (□); pApA_(Sp)*ApA (▪); pApApA_(Rp) *A (Δ); and pApApA_(Sp) *A (▴).

FIG. 2A represents the results of a ribosomal RNA cleavage assay with2',5'-phosphorothioate/phosphodiester trimer core 2-5A derivatives. Theprocedure was performed according to the method of Kariko et al.,Biochemistry 26: 7127-7135 (1987). 2-5A oligomer (phosphodiesterinternucleotide linkage) is also included for comparison. L929 cellextracts were incubated in the absence (lane 1) or presence of p₃ A₃ at10⁻⁸ M (lane 2), A_(Rp) *ApA at 10⁻⁶ M (lane 3), A_(Sp) *ApA at 10⁻⁶ M(lane 4), ApA_(Rp) *A at 10⁻⁶ M (lane 5), ApA_(Sp) *A at 10⁻⁶ M (lane 6)or A₃ at 10⁻⁶ M (lane 7). The positions of 28S and 18S rRNA are shown;the arrows indicate the positions of the well-characterized specificcleavage products (SCP) of RNase L.

FIG. 2B represents the results of a ribosomal RNA cleavage assayperformed according to the method of FIG. 2A with2',5'-phosphorothioate/phosphodiester trimer 5'-monophosphatederivatives. L929 cell extracts were incubated in the absence (lane 1)or presence of p₃ A₃ at 2×10⁻⁹ M (lane 2), pA₃ at 10⁻⁶ M (lane 3),pA_(Rp) *ApA at 10⁻⁷ M (lane 4), pA_(Sp) *ApA at 10⁻⁷ M (lane 5),pApA_(Rp) *A at 2×10⁻⁹ M (Lane 6), pApA_(Sp) *A at 10⁻⁷ M (lane 7).

FIG. 3A represents the results of a ribosomal RNA cleavage assayperformed according to the method of FIG. 2A with2',5'-phosphorothioate/phosphodiester tetramer core 2-5A derivatives.2-5A oligomer (phosphodiester internucleotide linkage) is also includedfor comparison. L929 cell extracts were incubated in the absence(lane 1) or presence of p₃ A₄ at 10⁻⁸ M (lane 2), A₄ at 10⁻⁵ M (lane 3),A_(Rp) *ApApA at 10⁻⁵ M (lane 4), A_(Sp) *ApApA at 10⁻⁵ M (lane 5),ApA_(Rp) *ApA at 10⁻⁵ M (lane 6), ApA_(Sp) *ApA at 10⁻⁵ M (lane 7),ApApA_(Rp) *A at 10⁻⁵ M (lane 8) or ApApA_(Sp) *A at 10⁻⁵ M (lane 9).

FIG. 3B represents the results of a ribosomal RNA cleavage assayperformed according to the method of FIG. 2A with2',5'-phosphorothioate/phosphodiester tetramer 5'-monophosphatederivatives. L929 cell extracts were incubated in the absence (lane 1)or presence of p₃ A₄ at 10⁻⁸ M (lane 2), pA₄ at 10⁻⁶ M (lane 3), pA_(Rp)*ApApA at 10⁻⁶ M (lane 4), pA_(Sp) *ApApA at 10⁻⁶ M (lane 5), pApA_(Rp)*ApA at 10⁻⁸ M (lane 6), pApA_(Sp) *ApA at 10⁻⁵ M (lane 7), pApApA_(Rp)*A at 10⁻⁷ M (lane 8) or pApApA_(Sp) *A at 10⁻⁷ M (lane 9).

FIG. 4A represents the results of a ribosomal cleavage assay performedaccording to the method of FIG. 2A, indicating the inhibition ofactivation of RNase L by pApA_(Sp) *A in L929 cell extracts and bypartially-purified RNase L. L929 cell extracts were incubated in thepresence of p₃ A₃ at 10⁻⁹ M (lanes 1 and 2), P₃ A₃ at 10⁻⁸ M (lanes 3and 4) pApA_(Rp) *A, at 10⁻⁹ M (lanes 5 and 6), pApA_(Rp) *A at 10⁻⁸ M(lanes 7 and 8), and pApA_(Sp) *A at 10⁻⁶ M (lanes 2, 4, 6 and 8).

FIG. 4B represents the results of a radiobinding assay indicating theactivation of RNase L partially-purified from L929 cell extracts.Activation of RNase L was determined by the conversion of poly(U) ³²P!pCp to acid-soluble fragments by immobilization on 2-5A₄core-cellulose. 100% represents 25,000 dpm of poly(U) 32P!pCp bound toglass fiber filters. 2-5 A oligomer (phosphodiester internucleotidelinkage) is also included for comparison. The curves are labeled asfollows: p₃ A₃ (); p₃ A₃ +pApA_(Sp) *A at 10⁻⁶ M (∘).

FIG. 5A represents the results of an assay performed according to themethod of Henderson et al., Virology 182: 186-198 (1991), indicating theinhibition of HIV-1 (IIIB)-induced syncytia formation by adenosine, 2-5Atrimer or tetramer core or 2',5'- phosphorothioate/phosphodiester trimerderivatives: A_(Rp) *ApA, A_(Sp) *ApA, ApA_(Rp) *A, ApA_(Sp) *A, A_(Rp)*ApApA, A_(Sp) *ApApA, ApA_(Rp) *ApA, ApA_(Sp) *ApA, ApApA_(Rp) *A,ApApA_(Sp) *A.

FIG. 5B is the results of an assay performed according to the method ofFIG. 5A with a 2',5'-phosphorothioate/phosphodiester tetramer derivativeof 2-5A core.

DETAILED DESCRIPTION OF THE INVENTION

The individual nucleotide linkages of the trimer and tetramerderivatives of 2',5'-oligoadenylate (2-5A) were stereochemicallymodified via phosphorothiate substitution by phosphotriester andphosphoramidite chemical synthesis. The approach described hereinutilizes fully protected monomeric building blocks (Schemes 1 and 2)below, which can be individually manipulated. The protecting groupsremain in place during the chemical synthesis of the oligonucleotidechain and are removed at the end of the sequence by β-elimination.

The phosphorothioate/phosphodiester trimer cores, A_(Rp) *ApA 10, A_(Sp)*ApA 11, ApA_(Rp) *A 23, and ApA_(Sp) *A 24, were chemically synthesizedand separated by preparative thin layer chromatography on silica gel,deblocked and purified by applying the residue on a DEAE Sephadexcolumn. The four trimer cores are prepared from phosphoramiditeintermediates 4 and 15. The synthesis relies on separation of fullyresolved protected intermediates, 8, 9, 21 and 22 followed by removal ofall blocking groups to yield the individually substituted2',5'-phosphorothioate/phosphodiester trimer adenylate cores. While notpart of the invention, the preparation of the dimer core 6 is includedfor completeness.

The selectively substituted tetramer cores, 38 and 39, 51 and 52 and 57and 58, were derived from the fully protected trimer cores, 30 and 31,and subsequently subjected to detrilylation and condensation to add thetetramer moiety.

The compounds of the present invention comprise 2-5A derivatives thatare (i) nuclease-resistant, (ii) non-toxic, (iii) able to activate orinactivate RNase L and (iv) able to inhibit HIV-1 replication. Theinventive compounds are chemically synthesizedphosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives inwhich at least one 2',5'-phosphodiester bond has been selectivelyreplaced with a 2',5'-phosphorothioate bond. The chemical synthesis ofthese phosphorothioate/phosphodiester derivatives utilizes thephosphotriester and phosphoramidite approach in which reactivefunctional groups are protected by blocking groups which can beindividually manipulated. The phosphorothioate/phosphodiester trimer andtetramer 2-5A derivatives reveal heretofore unknown aspects of thestereochemical requirements for activation of RNase L, namely, thatactivation of RNase L requires PR chirality in the secondinternucleotide linkage from the 5'-terminus of the 2-5A molecule andthat PS chirality in the second internucleotide linkage results in 2-5Aderivatives that are antagonists of RNase L activation. Without wishingto be bound by any theory, it appears that PR chirality in the secondinternucleotide linkage from the 5'-terminus may serve to facilitateformation of a productive complex between RNase L, the allostericactivator (ApA_(Rp) *A or ApA_(Rp) *ApA) and the RNA substrate such thathydrolysis of HIV-1 RNA can occur.

Phosphorothioate substitution of individual internucleotide linkages inthe 2-5A molecule has revealed that inhibition of HIV-1 replication isinfluenced by the location and stereoconfiguration of the chiralphosphorothioate group in the phosophorothioate/phosphodiesterderivatives. Of the four phosphorothioate/phosphodiester trimer corederivatives, ApA_(Rp) *A and ApA_(Sp) *A were the most efficientinhibitors of HIV-1 induced syncytia formation (FIG. 4A). Of the sixphosphorothioate/phosphodiester tetramer core derivatives, ApApA_(Rp) *Aand ApApA_(Sp) *A were the most efficient inhibitors (FIG. 4B). A_(Rp)*A, A_(Sp) *A, 3',5'-A₄, adenosine and adenine did not inhibit HIV-1 RTactivity. Whereas ApA_(Rp) *A and ApA_(Sp) *A are bothphosphodiesterase-resistant and inhibit HIV-1 RT, the ApA_(Rp) *Aenantiomer (but not the ApA_(Sp) *A enantiomer) can also activate RNaseL.

In this regard it appears, again, without wishing to be bound by anytheory, that the relative differences in the inhibition of HIV-1replication by the phosphorothioate/phospohodiester trimer and tetramercore derivatives may be explained by their resistance to hydrolysis byserum phosphodiesterases (see Table 1, infra.). In contrast to2',5'-phosphodiester bonds in authentic A₂ and A₃ which are totallyhydrolyzed in serum-containing medium in 20 minutes, both PR and PS2',5'-phosphorothioate bonds are more stable to hydrolysis byphosphodiesterases. The phosphorothioate/phosphodiester tetramer corederivatives which are stereochemically modified at the 5'-terminus(A_(Rp) *ApApA and A_(Sp) *ApApA) are rapidly hydrolyzed from the2',3'-terminus to their respective dimers, A_(Rp) *A and A_(Sp) *A.These dimers, although resistant to further hydrolysis, can neitheractivate RNase L nor inhibit HIV-1 replication. The remaining fourphosphorothioate/phosphodiester tetramer core derivatives (ApA_(Rp)*ApA, ApA_(Sp) *ApA ApApA_(Rp) *A and ApApA_(Sp) *A) are hydrolyzed fromthe 5'-terminus to form their respective trimer cores, A_(Rp) *ApA,A_(Sp) *ApA, ApA_(Rp) *A and ApA_(Sp) *A, respectively. Because ApA_(Rp)*A and ApA_(Sp) *A are efficient inhibitors of HIV-1 replication, thishydrolysis most likely accounts for the antiviral action of the tetramerderivatives, ApApA_(Rp) *A and ApApA_(Sp) *A. Therefore, the decreasedanti-HIV-1 activity observed with ApA_(Rp) *ApA and ApA_(Sp) *ApA(relative to ApApA_(Rp) *A and ApApAp_(Sp) *A) is likely due tohydrolysis from the 5'-terminus to form ApA_(Rp) *A and ApA_(Sp) *A,which are very efficient inhibitors of HIV-1 induced syncytia formation(compare FIGS. 4A and 4B).

In preliminary experiments, all phosphorothioate/phosphodiester trimerand tetramer 2-5A core derivatives of the present invention have beenshown to inhibit HIV-1 RT. Inhibition ranges from 22% to 70%. A_(Rp) *A,A_(Sp) *A, 3',5'-A₄, adenosine and adenine did not inhibit HIV-1 RTactivity. Whereas ApA_(Rp) *A and ApA_(Sp) *A are bothphosphodiesterase-resistant and inhibit HIV-1 RT, the ApA_(Rp) *Aenantiomer (but not the ApA_(Sp) *A enantiomer) can also activate RNaseL.

These three biological properties (i.e., resistance to hydrolysis byphospohodiesterases, inhibition of reverse transcriptase and activationof RNase L) may account for the 100% inhibition of HIV-1 replicationobserved with ApA_(Rp) *A.

The compounds of the invention are advantageously prepared as solublesalts of sodium, ammonium or potassium. The preparative scheme beginswith6-N-benzoyl-3'-O-tert-butyldimethylsilyl-5'-O-monomethoxy-trityladenosine1, which is advantageously prepared from adenosine according to theprocedure of Flockerzi et al., Liebig's Ann. Chem., 1568-1585 (1981).Preparation of the compounds of the present invention is illustrated inmore detail by reference to the following non-limiting examples.

3-Nitro-1,2,4-triazole and p-nitrophenylethanol used in the examples maybe prepared advantageously from published procedures: Chattopadhyaya etal., Nucleic Acids Res. 8:2039-2053 (1980); Schwarz et al., TetrahedronLett., 5513-5516 (1984); Uhlmann et al., Helv. Chim. Acta 64:1688-1703(1981). These compounds are also available commercially in the UnitedStates. 3-Nitro-1,2,4-triazole is available from Aldrich Chemical Co.,P.O. Box 355, Milwaukee, Wis. 53201 (1986-1987 cat. no. 24,179.2).p-Nitrophenylethanol is available from Fluka Chemical Corp. (cat. no.73,610).

Pyridine and triethylamine used in the examples were purified bydistillation over KOH, tosyl chloride and calcium hydride.Dichloromethane was distilled over calcium chloride and then passedthrough basic alumina. Pure acetonitrile was obtained by distillationover calcium hydride.

Purification of the protected nucleotides was achieved by preparativecolumn chromatography on silica gel 60 (0.063-0.2 mesh, Merck) and bypreparative thick layer chromatography on silica gel 60 PF₂₅₄ (Merck).Thin layer chromatography ("TLC") was carried out on precoated thinlayer sheets F 1500 LS 254 and cellulose thin layer sheets F 1440 fromSchleicher & Scheull.

Scheme 1 is the reaction scheme for the preparation of the fullyresolved trimers, having phosphorothioate substitution of the firstinternucleotide linkage A_(Rp) *ApA 10 and A_(Sp) *ApA 11, from theprotected intermediates, 8 and 9, wherein "bz" denotes the benzoylradical, "tbds" denotes the tert-butyldimethylsilyl radical, "ce"denotes the cyanoethyl radical, "npe" denotes the nitrophenylethoxyradical and "MeOTr" represents the monomethoxytrityl radical. Thepreparation of the trimer cores, 10 and 11 is set forth in Preparations1 through 4 and Example 1. Preparation 4 illustrates the fully protectedtrimer core, while Example 1 illustrates the removal of the blockinggroups and the chemical purification of the fully resolved isomers.##STR7##

PREPARATION 1 a. Bis-(diisopropylamino)-(β-cyanoethoxy)phosphane 3

Preparation of the titled compound was in accord with the procedure ofKraszewski & Norris, Nucleic Acids Research Sump. Ser. 18: 177-80(1987). β-Cyanoethanol (7 g; 0.1 mole) in absolute CH₃ CN (40 ml wasadded dropwise within 30 min to a solution of freshly distilled PCl₃ (40ml; 0.4 mole) at room temperature ("r.t.") and under nitrogenatmosphere. After stirring for 3.5 h, the solvent and excess PCl₃ wereremoved in high vacuum, the residue was dissolved in 450 ml of absoluteether and at -10° C. reacted with N,N-diisopropylamine (127 ml; 0.9mole) by dropwise addition within 1 h under nitrogen atmosphere. Thereaction mixture was stirred at -10° C. for 30 min and at r.t. for 15 h.The precipitate was filtered under nitrogen and the solvent was removedin vacuo. The yellow crude product was fractionally distilled over CaH₂to give 14.7 g (49%) of pure 3 of b.p. 114°-118° C. This reagent wasstored at -20° C. under nitrogen. ¹ H-NMR (CDCl₃): 3.75 (s, 2H, CH₂);3.52 (m, 4H, 4 N--CH); 2.60 (t, 2H, β-CH₂); 1.17+1.14 (2d, 24H, 4N--C(CH₃)₂). ³¹ P-NMR (CDCl₃): 124.6 ppm.

b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenosine-2'-O-(β-cyanoethyl)-N,N-diisopropylamino!-phosphoramidite 4

METHOD A

The preparation of the titled compound was in accord with the proceduresof Sinha et al., Nucleic Acids Res. 12: 4539-4557 (1984) whereincompound 1, Flockerzie et al. (1981), supra, (3.79 g; 5 mmole) anddiisopropyl-ethylamine (3.5 ml) were dissolved in dry CH₂ Cl₂ (20 ml)and chloro-N,N-diisopropylaminocyanoethoxy phosphane (2.37 g; 10 mmole)was added. After 1.5 h stirring under nitrogen at r.t., the reactionmixture was diluted with EtOAc (100 ml) and the organic phase was washedwith a saturated NaHCO₃ /NaCl solution (2×80 ml). The organic layer wasdried over Na₂ SO₄, filtered and evaporated to dryness. The residue wasdissolved in CH₂ Cl₂ (10 ml) and added dropwise to n-hexane (200 ml) at-60° C. The product was collected and evaporated to dryness in highvacuum for 8 h to give 4.3 g (89%) of a colorless amorphous solid.

METHOD B

Alternatively, the titled compound was prepared according to theprocedure of Kraszewski and Norris (1987), supra. In this methodcompound 1 (3.79 g; 5 mmole) and tetrazole (0.175 g; 2.5 mmole) weredissolved in dry CH₂ Cl₂ (20 ml) and thenbis-(diisopropylamino)-(β-cyanoethoxy)phosphane 3 (3 g; 10 mmole) wasadded. After stirring at r.t. under argon for 17 h, the reaction mixturewas extracted with EtOAc (100 ml) and washed with saturated NaHCO₃ /NaClsolution (80 ml). This was repeated twice and work-up was performedanalogous to method A to give 4.49 g (94%) of a colorless amorphouspowder. Anal. calc. for C₅₂ H₆₄ N₇ O₇ PSi×2 H₂ O (994.2): C 62.82, H6.89, N 9.86. Found: C 62.52, H 7.08, N 10.35. UV (MeOH): λ_(max) (logε)279 nm (4.33); 229 nm (4.43). R_(f) on silica gel with toluol/EtOAc(1/1, v/v): 0.64, 0.61 (diastereomers). ³¹ P-NMR (CDCl₃): 150.98,151.34.

PREPARATION 2

6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- O^(P)-(2-cyanoethyl)-5'-!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 6

The phosphoramidite 4 (2.88 g; 3 mmole) and 6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!adenosine 5, Flockerzie et al. (1981), supra,(1.2 g; 2 mmole) were dried at r.t. in high vacuum for 24 h anddissolved in dry CH₂ Cl₂ (30 ml). Tetrazole (0.5 g; 8 mmole) was addedand after 3 h stirring at r.t. under argon, a solution of I₂ 0.5 g H₂O/pyridine/CH₂ Cl₂ (1/3/1, v/v/v)! was added dropwise until the browncolor does not disappear. The mixture was stirred for 15 min, thendiluted with CHCl₃ (300 ml). The organic phase was saturated with Na₂ S₂O₃ /NaCl (3×80 ml), dried over Na₂ SO₄ and evaporated to dryness. Finalcoevaporation was done with toluene (3×20 ml). The crude product waspurified by silica gel column chromatography (15×2.5 cm) using CHCl₃(100 ml), CHCl₃ /MeOH (100/0.5, v/v; 1.5 L) and CHCl₃ /MeOH: (100/1,v/v) to elute the product. Product fractions were collected andevaporated to dryness to give 2.33 g (79%) of the dimer 6 in the form ofa solid foam. Anal. calc. for C₇₅ H₉₄ N₁₁ O₁₃ PSi₃ (1490.9): C 60.42, H6.49, N 10.33. Found: C 60.50, H 6.49, N 10.22. UV (MeOH): λ_(max)(logε) 278 nm (4.62), 230 nm (4.62). R_(f) on silica gel with CHCl₃/MeOH (95/5, v/v)=0.56. ³¹ P-NMR (CDCl₃): -0.74 and -1.07 ppm(diastereomers).

PREPARATION 3 6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-adenylyl-2'-O^(P) -(2-cyanoethyl)-5'!-6-N-benzoyl-2',3'-O-di-(tert-butyl)dimethylsilyl!adenosine 7

Compound 6 (2.22 g; 1.51 mmole) was stirred with 2% p-TsOH in CH₂ Cl₂/MeOH (4/1, v/v; 30 ml) at r.t. for 30 min. The reaction mixture wasdiluted with CH₂ Cl₂ (300 ml), washed with phosphate buffer, pH 7.0(2×100 ml), dried over Na₂ SO₄ and evaporated to dryness. The residuewas applied to a silica gel column (9×4.5 cm), washed with CHCl₃ (0.7 L)and CHCl₃ /MeOH (100/1, v/v; 300 ml). The product was eluted with CHCl₃/MeOH (50/1, v/v; 300 ml and 100/3, v/v; 300 ml). The combined productfractions were evaporated to dryness in high vacuum to give 11.65 g(90%) of 5'-hydroxy dimer 7 as an amorphous solid. Anal. calc. for C₅₅H₇₈ N₁₁ O₁₂ PSi₃ ×H₂ O (1218.5): C 54.21, H 6.62, N 12.64. Found: C54.53, H 6.58, N 12.62. UV (MeOH): λ_(max) (logε) 278 nm (4.60), 232 nm(4.42.). R_(f) on silica gel with CHCl₃ /MeOH (95/5, v/v)=0.36. ³¹ P-NMR(CDCl₃): -0.77 and -1.30 ppm (diastereomers).

PREPARATION 4 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR)-thioadenylyl-2'-O^(P) -(2-cyanoethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- O^(P)-(2-cyanoethyl)-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A_(Rp) *ApA 8 b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PS)-thioadenylyl-2'-O^(P) -(2-cyanoethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- O^(P)-(2-cyanoethyl)-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A_(Sp) *ApA 9

The phosphoramidite 4 (2.79 g; 2.92 mmole), the 5'-hydroxy dimer 7 (1.94g; 1.62 mmole) and tetrazole (0.567 g; 8.1 mmole) were dissolved in dryCH₃ CN (8.1 ml) and stirred at r.t. under nitrogen. After 3 h, S₈ (1.66;6.48 mmole) and pyridine (7.8 ml) were added and stirred further for 20h at r.t. The reaction mixture was then diluted with CH₂ Cl₂ (300 ml),washed with saturated NaCl (2×200 ml), dried over Na₂ SO₄ and evaporatedto dryness. Final coevaporation was with toluene (3×20 ml). The crudediastereomeric mixture (A_(Rp) *ApA 8+A_(Sp) *ApA 9) was dissolved inCH₂ Cl₂ and applied to a silica gel column (21×3.5 cm). The column waswashed with CH₂ Cl₂ (450 ml) and CH₂ Cl₂ /MeOH (99/1, v/v; 200 ml) andthe product was eluted with CH₂ Cl₂ /MeOH (97/3, v/v; 400 ml). Theproduct fractions were collected and evaporated to dryness to give 3.16g (93%) of an isomeric mixture of A_(Rp) *ApA 8 and A_(Sp) *ApA 9.Separation into the pure diastereoisomers was achieved by mediumpressure chromatography as described above by elution with CHCl₃ /MeOH(99/1, v/v; 800 ml; 20 ml/fraction; fractions 1-40) followed by elutionwith CHCl₃ /MeOH (95/5, v/v; 800 ml; 20 ml/fraction; fractions 41-80).The fully protected A_(Rp) *ApA isomer 8 (0.287 g) was eluted infractions 21-56 (20 ml/fraction). Fractions 57-61 gave the isomermixture (0.07 g) and the fully protected A_(Sp) *ApA isomer 9 (0.132 g)was eluted in fractions 62-64. Chromatographic separation was repeatedwith each 0.5 g of the crude mixture to yield 1.62 g (51%) of A_(Rp)*ApA 8 and 0.94 g (30%) of A_(Sp) *ApA 9. Anal. calc. for A_(Rp)*ApA-C₁₀₁ H₁₂₇ N₁₇ O₁₉ P₂ SSi₄ (2089.6): C 58.05, H 6.13, H 11.40.Found: C 58.65, H 6.24, N 11.50. UV (MeOH): λ_(max) (logε) 279 nm(4.76), 260 nm (4.56), 236 nm (4.73). R_(f) on silica gel with CHCl₃/MeOH (97/3, v/v)=0.35. ³¹ P-NMR (CDCl₃): 69.35 and -1.10 ppm. Anal.calc. for A_(Sp) *ApA-C₁₀₁ H₁₂₇ N₁₇ O₁₉ P₂ SSi₄ (2089.6): C 58.05, H6.13, N 11.25. Found: C 57.03, H 6.33, N 11.14. UV (MeOH): λ_(max)(logε) 279 nm (4.77), 260 nm (4.57), 236 nm (4.73). ³¹ P-NMR (CDCl₃):68.33 and -0.84 ppm.

EXAMPLE 1 a. (PR)-P-Thioadenylyl-2'-5'-adenylyl-2'-5'-adenosine A_(Rp)*ApA 10 b. (PS)-P-Thioadenylyl-2'-5'-adenylyl-2'-5'-adenosine A_(Sp)*ApA 11

The corresponding fully protected trimers 8 and 9, respectively, wereseparately deblocked by stirring the trimer (0.06 g; 0.029 mmole) with2% p-TsOH in CH₂ Cl₂ /MeOH (4/1, v/v; 1.2 ml) for 1.5 h at r.t. Thereaction mixture was diluted with CHCl₃ (50 ml), washed with H₂ O (2×25ml), dried and evaporated to dryness. The crude product was purified onpreparative silica gel plates (20×20×0.2 cm) in CHCl₃ /MeOH (8/2, v/v).The product bands were eluted with CHCl₃ /MeOH (4/1, v/v) and evaporatedto a foam to give 0.04 g (84%) of the A_(Rp) *ApA isomer 10 and 0.034 g(73%) of the A_(Sp) *ApA isomer 11. The 5'-hydroxy trimer (0.034 g; 0.08mmole) was then stirred with 0.5M DBU in pyridine (5.0 ml) and afterstirring at r.t. for 20 h, the solution was neutralized with 1M aceticacid in dry pyridine (2.5 ml) and evaporated to dryness. The residue wastreated with methanolic ammonia (5 ml) and after 48 h stirring thesolvents were removed in vacuo. Desilylation was performed with 1Mtetrabutylammonium fluoride in THF (2 ml). After 48 h stirring, thesolvent was removed in vacuo and the residue was dissolved in H₂ O (10ml) and applied to a DEAE Sephadex A-25 column (60×1 cm). The pureproduct was eluted with a linear gradient of 0.14-0.17M TEAB buffer, pH7.5. After evaporation and coevaporation with water several times, thetrimer was applied to four paper sheets (35×50 cm) and developed ini-PrOH/conc. ammonia/H₂ O (6/1/3, v/v/v). The product band was cut out,eluted with H₂ O, evaporated and lyophilized to give 500 O.D.₂₆₀ nmunits (79%) of the A_(Rp) *ApA isomer 10 and 410 O.D.₂₆₀ nm units (65%)of the A_(Sp) *ApA isomer 11. UV λ_(max) in both cases was 258 nm in H₂O. A_(Rp) *ApA 10: R_(f) on cellulose in i-PrOH/ammonia/H₂ O (6/1/3,v/v/v)=0.33. ¹ H-NMR (D₂ O): 8.20; 8.19; 8.14 (3s, 3H, H--C(8)); 7.97(1s, 2H, 2 H--C(2)) and 7.76 (1 s, 1H, 1 H--C(2)); 6.08; 5.93; 5.82 (3d,3H, 3 H--C(1')). Retention time on reverse-phase HPLC was 5.60 min.A_(Sp) *ApA 11: R_(f) on cellulose in i-PrOH/ammonia/H₂ O (6/1/3,v/v/v)=0.33. ¹ H-NMR (D₂ O): 8.14; 8.09; 8.02 (3s, 3H, H--C(8)); 7.94;7.89: 7.80 (3s, 3H, 2 H--C(2)); 6.03; 5.92; 5.80 (3d, 3H, 3 H--C(1')).Retention time on reverse-phase HPLC was 6.51 min.

Scheme 2 is the reaction scheme for the preparation of the remainingpair of trimer cores, ApA_(Rp) *A 23 and ApA_(Sp) *A 24, from theprotected intermediates 21 and 22, and is outlined in detail inpreparations 5 and 6 and Example 2, below. ##STR8##

PREPARATION 5 a. N,N-Diisopropyl-trimethylsilylamine

The preparation of the titled compound was in accord with the procedureof Noth and Staudigl, Chem. Ber. 115: 3011-3024 (1982). Methyl iodide(37.6 ml; 0.6 mole) in absolute ether (50 ml) was added dropwise (over90 min) to a suspension of 14.6 g (0.6 mole) of magnesium and a fewcrystals of iodine in absolute ether (100 ml). The reaction was thenstirred for 30 min until all the magnesium was dissolved. Subsequently,N,N-diisopropylamine (78 ml; 0.55 mole) was added within 10-15 min andthe reaction was refluxed for 1 h. After cooling to 0° C.,trimethylsilyl chloride (76 ml; 0.6 mole) was added dropwise and thereaction mixture was again heated in an oil-bath with vigorous stirringto 80° C. for 20 h. The supernatant liquid was decanted and the residuewas extracted with ether (4×50 ml). The supernatant and the etherextract were combined. The solvent, excess trimethylsilyl chloride andunreacted N,N-diisoproylamine were removed by distillation. The productwas then isolated by distillation under vacuum at an oil-bathtemperature of 60° C. to yield 73 g (80%), Kp₁₈ =36°-39° C. ¹ H-NMR(CDCl₃): 0.08 (s, 9H, SiCH₃); 1.04-1.07 (d, 12H, N--C--CH₃), 3.2 (m, 2H,N--CH).

b. Chloro-N,N-diisopropylamino-2-(4-nitrophenyl)ethoxy-phosphane 14

p-Nitrophenylethanol (4.16 g; 25 mmole) was added portion wise to asolution of freshly distilled PCl₃ (14 ml; 0.16 mole) in absolute ether(40 ml) at -30° C. under a nitrogen atmosphere within 45 min. Thereaction mixture was stirred at r.t. for 1.5 h and the solvent andexcess PCl₃ were then removed in vacuo at 0° C. The residue was treatedwith N,N-diisopropyl-trimethylsilylamine (Preparation 5a) (4.33 g; 25mmole) at 0° C. under a nitrogen atmosphere for 30 min and then at r.t.for 20 h. The resulting trimethylsilyl chloride was removed under highvacuum at r.t. to yield a syrupy pale yellow product (7.1 g; 85%) whichcrystallized upon storage at -20° C. This material was then used for thesubsequent phosphitylation reactions. ¹ H-NMR (CDCl₃): 8.1-8.2 (m, 2H, oto NO₂); 7.39-7.43 (m, 2H, m to NO₂); 4.04-4.18 (m, 2H, P--O--CH₂);3.63-3.79 (m, 2H, N--CH); 3.07-3.13 (t, 2H, P--O--C--CH₂); 1.14-1.27(2d, 12H, N--C--CH₃). ³¹ P-NMR (CDCl₃): 181.60 ppm.

c. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenosine-2'-O-(4-nitrophenyl)ethyl)-N,N-diisopropylaimino!-phosphoramidite 15

METHOD A

Compound 1 (3.79 g; 5 mmole) and diisopropylethylamine (3.5 ml) weredissolved in dry CH₂ Cl₂ (20 ml) and thenchloro-N,N-diisopropylamino-2-(4-nitrophenyl)-ethoxyphosphane 14 (2.37g; 10 mmole) was added dropwise under a nitrogen atmosphere. Afterstirring at r.t. for 2 h, the reaction mixture was diluted with EtOAc(200 ml), the organic phase was washed with a saturated NaHCO₃ /NaClsolution (3×80 ml), dried over Na₂ SO₄ and evaporated to dryness. Thecrude product was dissolved in toluene/EtOAc (7/3, v/v) andchromatographed on a silica gel column (12×2 cm) equilibrated withEtOAc/NEt₃ (95/5, v/v). The product fractions were eluted withEtOAc/NEt₃ (95/5, v/v), collected and evaporated to dryness, yielding 15(5.28 g; 79%) as a colorless solid foam. Anal. calc. for C₅₇ H₆₈ N₇ O₉PSi (1054.3): C 64.94, H 6.50, N 9.30. Found: C 64.81, H 6.51, N 9.01.UV (MeOH): λ_(max) (logε) 277 nm (4.50), 229 nm (4.48). ³¹ P-NMR(CDCl₃): 150.27, 150.01 ppm. R_(f) on silica gel in toluene/EtoAC (1/1,v/v): 0.62 and 0.68 (diastereomers).

METHOD B

Alternatively, compound 15 was synthesized using bis-(diisopropylamino)-2-(4-nitrophenyl)ethoxy!-phosphane 27, infra. To a solution of 1.52 g (2mmole) of compound 1 in absolute CH₃ CN (10 ml), bis-(diisopropylamino)-2-(4-nitrophenyl)ethoxy!-phosphane 27 (1.59 g, 4 mmole) and tetrazole(0.07 g; 1 mmole) were added under nitrogen atmosphere and the reactionmixture was stirred for 17 h at r.t. The reaction mixture was dilutedwith EtOAc (120 ml) and washed twice with saturated NaHCO₃ /NaClsolution (60 ml), dried over Na₂ SO₄ and evaporated to dryness. Thecrude solid foam was applied onto a flash silica gel column (20×2.5 cm)and chromatographed with toluene/EtOAc (1/1, v/v; 250 ml). The productfraction (90 ml) was evaporated to give 15 (1.9 g, 90%) as a colorlesssolid foam.

c. Bis-(diisopropylamino)- 2-(4-nitrophenyl)ethoxy!-phosphane 27

2-(4-Nitrophenyl)ethanol (8.35 g, 50 mmole) was added in small portionsover 30 min to a solution of distilled PCl₃ (28 ml; 280 mmole) inabsolute ether (80 ml) at -5° C. under a nitrogen atmosphere. Afterstirring for 15 min at -5° C. and 1.5 h at r.t., the solvent and excessPCl₃ were removed under high vacuum. Then, the yellowish syrupy residuewas dissolved in 200 ml of absolute ether and reacted at -10° C. withN,N-diisopropylamine (64 ml, 450 mmole) by dropwise addition over 30 minunder a nitrogen atmosphere. The reaction mixture was stirred at -10° C.for 15 min and r.t. for 16 h. The voluminous precipitate ofN,N-diisopropyl-amine hydrochloride was filtered under nitrogen and thesolvent was removed in vacuo. The yellowish syrupy product (17.6 g;89%), which crystallized on storage at -20° C. was pure enough to beused for phosphitylation reactions. ¹ -NMR (CDCl₃): 8.10-8.13 (d, 2H, oto NO₂); 7.36-7.40 (d, 2H, m to NO₂); 3.75-3.82 (q, 2H, P--O--CH₂);3.36-3.51 (m, 2H, N--CH); 2.95-3.00 (t, 2H, P--O--C--CH₂); 1.05-1.12(2d, 12H, N--C--CH₃). ³ P-NMR (CDCl₃): 123.53 ppm.

PREPARATION 6 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'- (O^(P)-2-(4-nitro-phenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine ApA_(Rp) *A 21 b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'- (O^(P)-2-(4-nitro-phenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine ApA_(Sp) *A 22

Triethylammonium 6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)adenosine-2'-2-(4-nitrophenyl)ethyl!-phosphate 20 (0.10 g; 0.1 mmole), Charubala etal., Liebig's Ann. Chem., 2392-2406 (1981), and the respective5'-hydroxy dimers PR 18 and PS 19, (0.066 g; 0.05 mmole), Charubala andPfleiderer (1992) supra, were coevaporated with dry pyridine (3×5 ml),dissolved in one ml dry pyridine and (2,4,6-triisopropyl)benzenesulfonylchloride (0.062 g; 0.2 mmole) and 3-nitro-1,2,4-triazole (0.068 g; 0.6mmole), Kroger and Mietchen, Z. Chem. 9: 378-379 (1969); Jones et al.,Tetrahedron 36: 3075-3085 (1980), were added. After stirring at r.t. for20 h, the reaction mixture was diluted with CHCl₃ (100 ml), washed withH₂ O (2×50 ml), dried and evaporated. Final evaporations were done withtoluene (2×10 ml) to remove pyridine. The crude trimers 21 and 22,respectively, were purified by silica gel column chromatography (15×2cm), using first CHCl₃ and then CHCl₃ /MeOH (100/1, v/v) as eluants. Theproduct fraction was collected and evaporated to a solid foam, which wasdried under high vacuum to give 0.08 g (70%) of 21. Anal. calc. for C₁₁₁H₁₃₅ N₁₇ O₂₃ P₂ SSi₄ ×2 H₂ O (2317.8): C 57.52, H 5.95, N 10.27. Found:C 57.15, H 6.13, N 10.72. UV (MeOH): λ_(max) (logε) 276 nm (4.87), 227nm (4.83). R_(f) on silica gel in CH₂ Cl₂ /EtOAc (1/1)=0.63. ³¹ P-NMR(CDCl₃): 69.88 and -1.0 ppm.

EXAMPLE 2 a. Adenylyl-(2'-5')-(PR)-P-thioadenylyl-(2'-5')-adenosineApA_(Rp) *A 23 b. Adenylyl-(2'-5')-(PS)-P-thioadenylyl-(2'-5')-adenosineand ApA_(Sp) *A 24

The fully protected trimers, ApA_(Rp) *A 21 and ApA_(Sp) *A 22, wereseparately deblocked by stirring the corresponding trimer (0.088 g;0.037 mmole) with 2% p-TsOH in CH₂ Cl₂ /MeOH (4/1, v/v; 0.8 ml. After 30min stirring at r.t., the reaction mixture was diluted with CHCl₃ (50ml) and washed with H₂ O (2×25 ml). The organic phase was dried overNaSO₄ and evaporated to dryness. The crude product was purified or asilica gel column (5×2 cm) and the product eluted with CHCl₃ /MeOH(100/1, v/v), evaporated and dried under high vacuum to give 0.073 g(94%) of the 5'-hydroxy trimer ApA_(Rp) *A 21 and 0.061 g (84%) of the5'-hydroxy trimer ApA_(Sp) *A 22. The resulting 5'-hydroxy trimer (0.04g; 0.02 mmole) was then stirred with 10 ml of 0.5M DBU in pyridine.After 24 h, the solution was neutralized with 1M acetic acid in pyridine(10 ml) and evaporated to dryness. The residue was treated withsaturated methanolic ammonia (6 ml) and after stirring at r.t. for 48 h,the solvent was removed in vacuo and the residue was desilylated with 1MBu₄ NF in THF (5 ml) for 48 h. The solvent was then removed in vacuo andthe residue was dissolved in water (10 ml) and applied onto a DEAESephadex A-25 column (60×1 cm). The product was eluted with a lineargradient of 0.14-0.17M TEAB buffer, pH 7.5. After evaporation andcoevaporation with water several times, the trimer was applied to fourpaper sheets (35×50 cm) and developed in i-PrOH/conc. ammonia/H₂ O(6/1/3, v/v/v). The product band was cut out, eluted with H₂ O,evaporated and lyophilized to give 354 O.D.₂₆₀ nm units (79%) of theApA_(Rp) *A isomer 23 and 410 O.D.₂₆₀ nm units (58%) of the ApA_(Sp) *Aisomer 24. UV λ_(max) in both cases was 258 nm in H₂ O. ApA_(Rp) *A 23:R_(f) on cellulose in iPrOH/ammonia/H₂ O (6/1/3, v/v/v) 0.34. ¹ H-NMR(D₂ O): 8.17; 8.16; 8.09 (3s, 3H, H--C(8)); 7.90, 7.78 (2 s, 3H,3×H--C(2)); 6.04; 5.96; 5.80 (3d, 3H, 3 H--C(1')). Retention time onreverse-phase HPLC was 5.98 min. ApA_(Sp) *A (24): R_(f) on cellulose ini-PrOH/ammonia/H₂ O (6/1/3, v/v/v)=0.33. ¹ H-NMR (D₂ O): 8.17; 8.07;8.04 (3s, 3H, 3×H--C(8)); 8.01; 7.92: 7.72 (3s, 3H, 2 3×H--C(2)); 6.04;5.92; 5.82 (3d, 3H, 3×H--C(1 )). Retention time on reverse-phase HPLCwas 7.23 min.

Preparations 7 and 8 begin the preparation for the fully resolvedtetramers, ApA_(Rp) *ApA 38 and ApA_(Sp) *ApA 39, from theircorresponding dimer 28 (Scheme 1). The reaction scheme continues withthe addition of the trimer moiety in Preparations 9 and 10 (Scheme 2).

PREPARATION 7 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-(monomethoxytrityl)-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A.sub.(Rp,Sp) *A 28

The phosphoramidite 15 (1.41 g; 1.34 mmole), 6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5 (0.36 g, 0.93 mmole) andtetrazole (0.188 g, 2.68 mmole) were stirred at r.t. in absolute CH₃ CN(9 ml) under a nitrogen atmosophere. After 4 h, a solution of I₂ 0.5 gin CH₂ Cl₂ /H₂ O/pyridine (1/1/3, v/v/v)! was added dropwise until thebrown color did not disappear. The mixture was stirred was stirred foranother 15 min, then extracted with CH₂ Cl₂ (3×60 ml) and saturated Na₂S₂ O₃ /NaCl solution (2×60 ml). The CH₂ Cl₂ phase was collected, driedover Na₂ SO₄, evaporated and coevaporated with toluene (2×20 ml) toremove the pyridine. The crude dimer (1.85 g) was dissolved in CH₂ Cl₂and applied onto a flash silica gel column (12×2.5 cm) andchromatographed using CH₂ Cl₂ /1% MeOH (400 ml), 2% MeOH (200 ml) and 3%MeOH (200 ml) to elute the product (600 ml). This fraction wasevaporated to dryness to give 1.45 g (quant. yield) of the dimer 28 as acolorless amorphous solid. The identity of the isolated dimer 28 wasproven by comparison with authentic material by spectrophotometriccomparison. The authentic material was synthesized by thephosphotriester method: Anal. calc. for ApA 28=C₈₀ H₉₈ N₁₁ O₁₅ PSi₃(1569.0): C 61.24, H 6.30, N 9.82. Found: C 61.24, H 6.24, N 9.65. UV(MeOH): λ_(max) (logε) 277 (4.69); 260 (4.54)!; 231 (4.66)!. R_(f) onsilica gel with CHCl₃ /MeOH (49/1, v/v)=0.37.

PREPARATION 8 6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-adenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine-5'-OH-A.sub.(Rp,Sp) *A 29

The crude dimer mixture 28 (2.24 g, 1.43 mmole) was stirred with 2%p-TsOH in CH₂ Cl₂ /MeOH (4/1, v/v, 20 ml) at r.t. for 30 min. Thereaction mixture was diluted with CH₂ Cl₂ (200 ml), washed with H₂ O(2×80 ml), dried over Na₂ SO₄ and evaporated to dryness. The colorlessamorphous residue (2.0 g) was applied onto a flash silica gel column(21×2.5 cm) and chromatographed with CH₂ Cl₂ (200 ml), CH₂ Cl₂ /2% MeOH(400 ml) and the product was eluted with CH₂ Cl₂ /2% MeOH (500 ml). Theproduct fraction was evaporated and dried under high vacuum to give 1.2g (75% calculated to compound 5 over 2 steps) of 5'-OH dimer 29 as anamorphous solid. The identity of the isolated dimer 29 with authenticmaterial was proven by chromatographic and spectrophotometriccomparison. Anal. calc. for 5'-OH-ApA 29=C₆₀ H₈₂ N₁₁ O₁₄ PSi₃ (1296.6):C 55.58, H 6.37, N 11.88. Found: C 55.33, H 6.38, N 11.78. UV (MeOH):λ_(max) (logε) 278 (4.68); 259 (4.51)!; 233 (4.46). R_(f) on silica gelwith toluene/EtOAc/MeOH (5:4:1)=0.53. ³¹ P=NMR (CDCl₃ : -0.36 and -0.73ppm.

PREPARATION 9 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'-!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A_(Rp) *ApA 30 b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PS)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'-!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A_(Sp) *ApA 31

The phosphoramidite 15 (0.59 g, 0.56 mmole), the 5'-hydroxy ApA dimer 29(0.52 g, 0.40 mmole) and tetrazole (0.079 g; 1.12 mmole) were dissolvedin dry CH₃ CN (4 ml) and stirred at r.t. under a nitrogen atmosphere.After 3 h, phosphoramidite 15 (0.464, 0.44 mmole) and tetrazole (0.062g, 0.88 mmole) were added again and the mixture was stirred for another3 h. Then, oxidation with S₈ (0.257 g, 1 mmole) and pyridine (2.6 ml)was followed within 16 h at r.t. The reaction mixture was diluted withCH₂ Cl₂ (200 ml) at r.t. The reaction mixture was diluted with CH₂ Cl₂(200 ml), washed with a saturated NaCl solution (2×80 ml), dried overNa₂ SO₄ and evaporated to dryness. Final coevaporation was done withtoluene (3×20 ml) to remove pyridine. The crude diastereoisomericmixture A(_(Rp),Sp)*ApA 30+31 was dissolved in CH₂ Cl₂ (20 ml), appliedonto a flash silica gel column (11×2.5 cm) and chromatographed with CH₂Cl₂ (400 ml), CH₂ Cl₂ /0.5% MeOH (200 ml), 1% MeOH (200 ml) and theproduct was eluted with CH₂ Cl₂ /1.5% MeOH (200 ml). The productfraction was evaporated to dryness to give 0.713 g (78%) of the isomericmixture 30+31. Separation into the pure diastereomers was achieved byapplication to preparative silica gel plates (40×20×0.2 cm, 8 plates) intoluene/EtOAc (1/1, v/v, 4 developments). The isomeric products bandswere separately eluted with CH₂ Cl₂ /MeOH (4/1, v/v) and evaporated tosolid foams, which were dried under high vacuum to give 0.311 g (34%) ofthe fully protected A_(Rp) *ApA isomer 30 and 0.245 g (270%) of thefully protected A_(Sp) *ApA isomer 31. Anal. calc. for A_(Rp) *ApA30=Cl₁₁₁ H₁₃₋₅ N₁₇ O₂₃ P₂ SSi₄ ×H₂ O (2299.8): C 57.97, H 6.00, N 10.35.Found: C 57.63, H 6.11, N 10.39. UV (MeOH): λ_(max) (logε) 278 (4.87);260 (4.72)!; 231. (4.75)!. R_(f) on silica gel with toluene/EtOAc (1/1,v/v, 2 developments) and toluene/EtOAc (1/2, v/v, 1 development)=0.37.Anal. calc. for A_(Sp) *ApA 31=C₁₁₁ H₁₃₅ N₁₇ O₂₃ P₂ SSi₄ ×2 H₂ O(2317.8): C 57.52, H 6.05, N 10.27. Found: C 57.44, H 6.19, N 10.41. UV(MeOH): λ_(max) (logε) 277 (4.86); 260 (4.72)!; 231 (4.75)!. R_(f) onsilica gel with toluene/EtOAc (1:1, 2 developments) and toluene/EtOAc(1:2, 1 development)=0.27.

PREPARATION 10 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'- O.sup.P-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5'-hydroxy A_(Rp) *ApA 32 b.6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'-O.sup.P -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5'-hydroxy A_(Sp) *ApA 33

The corresponding fully protected trimers 30 and 31, respectively, wereseparately detritylated by stirring the trimer (A_(Rp) *ApA 30: 0.263 g,0.115 mmole; A_(Sp) *ApA 31: 0.21 g, 0.92 mmole) with 2% p-TsOH in CH₂Cl₂ /MeOH (4/1, v/v, for 30: 3.2 ml; for 31: 2.6 ml) for 75 min at r.t.The reaction mixture was diluted with Ch₂ Cl₂ (120 ml), washed with H₂ O(2×40 ml), dried over Na₂ SO₄ and evaporated to dryness. The crudeproduct was purified on preparative silica gel plates (40×20×0.2 cm) intoluene/EtOAc (3/7, v/v), the product bands were eluted with CH_(2C1)2/MeOH (4/1, v/v) and evaporated to a solid foam to give 0.2 g (86%) ofthe 5'-hydroxy A_(Rp) *ApA 32 and 0.121 g (66%) of the 5'-hydroxy A_(Sp)*ApA 33, respectively. Anal. Calc. for 5'-OH-A_(Rp) *ApA 32=C₉₁ H₁₁₉ N₁₇O₂₂ SSI₄ (2009.4: C 54.39, H 5.97, N 11.85. Found: C 54.12, H 6.13, N1174. UV (MeOH): λ_(max) (logε) 278 (4.86); 260 (4.71)!; 233 (4.64!.R_(f) on silica gel with toluene/EtOAc (3:7, 2 developments)=0.35(diastereomers). ³¹ P-NMR: 69.60, 68.91, -0.36 and -0.56 ppm(diastereomers). Anal. calc. for 5'-OH-A_(Sp) *ApA 33=C₉₁ H₁₁₉ N₁₇ O₂₂P₂ SSi₄ (2009.4): C 54.39, H 5.97, N 11.85. Found: C 54.29, H 6.23, N11.51. UV (MeOH); λ_(max) (logε) 277 (4.85; 260 (4.71)!; 233 (4.63)!.R_(f) silica gel with toluene/EtOAc (3:7, 2 developments=0.42(diastereomers). ³¹ P-NMR: 69.28, 69.09, -0.33 and -0.56 ppm(diastereomers).

Scheme 3 is the reaction scheme for the preparation of the fullyresolved tetramers, ApA_(Rp) *ApA 38 and ApA_(Sp) *ApA 39, from theprotected intermediates, 34 and 35. The preparation of these compoundsis outlined in Preparations 11 and 12 and Example 3. ##STR9##

PREPARATION 11 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'- O^(P)-(2-(4-nitrophenyl)ethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- O^(P)-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl-adenosine ApA_(Rp) *ApA 34 b.6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'- O^(P)-(2-(4-nitrophenyl)ethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- O^(P)-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl-adenosineApA_(Sp) *ApA 35

The condensation to the fully protected tetramers 34 and 35,respectively, were separately realized by coevaporatingtriethylammonium-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)adenosine-2'-2-(4-nitrophenyl)ethyl!-phosphate 20 (0.108 g, 0.1 mmole) and the5'-hydroxy PR trimer 32 or PS trimer 33 (0.1 g, 0.05 mmole),respectively, with dry pyridine (3×2 ml), dissolved in dry pyridine (0.5ml) and then (2,4,6-triisopropyl)benzenesulfonyl chloride (0.061 g, 0.2mmole) and 3-nitro-1,2,4-triazole (0.068 g, 0.6 mmole) were added. Thesolution was stirred at r.t. for 21 h, then extracted with CH₂ Cl₂ (2×20ml) and H₂ O (3×20 ml). The organic phase was collected, dried over Na₂SO₄, evaporated and coevaporated with toluene (3×20 ml) to removepyridine. The crude tetramers 34 and 35, respectively, were separatelypurified on preparative silica gel plates (40×20×0.2 cm) withtoluene/EtOAc/MeOH (5/4/0.5, v/v/v/), the product bands were eluted withCH₂ Cl₂ /MeOH (4/1, v/v/) and evaporated to solid foams, which weredried under high vacuum to give 0.116 g (78%) of ApA_(Rp) *ApA 34 and0.12 g (8%) of the ApA_(Sp) *ApA 35. Anal. calc. for ApA_(Rp) *ApA34=C₁₄₂ H₁₇₂ N₂₃ O₃₂ P₃ SSi₅ ×2 H₂ O (3014.5): C 56.58, H 5.89, N 10.69;found: C 56.22, H 6.07, N 10.57. UV (MeOH): λ_(max) (logε) 277 (4.99):260 (4.85)!; 231 (4.85)!. R_(f) silica gel with toluene/EtOAc/MeOH(5:4:0.5)=0.63 (diastereomers). Anal. calc. for ApA_(Sp) *ApA 35=C₁₄₂H₁₇₂ N₂₃ O₃₂ P₃ SSi₅ ×H₂ O (2996.5): C 56.92, H 5.85, N 10.75; found: C56.40, H 5.89, N 10.61. UV (MeOH): λ_(max) (logε) 277 (5.01): 260(4.88)!; 232 (4.89)!. R_(f) silica gel with toluene/EtOAc/MeOH (5/4/0.5,v/v/v)=0.62 (diastereomers).

PREPARATION 12 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5'-OH-ApA_(Rp) *ApA 36 b.6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5'-OH-ApA_(Sp) *ApA 37

The fully protected tetramers (0.105 g; 0.035 mmole) 34 and 35,respectively, were separately detritylated by treatment with 20% p-TsOHin CH₂ Cl₂ /MeOH (4/1, v/v, 1.2 ml) for 1 h at r.t. The reaction mixturewas extracted with CH₂ Cl₂ (3×40 ml) and washed with H₂ O (3×30 ml). Theorganic phase was collected, dried over Na₂ SO₄ and evaporated todryness. The resulting residue was purified on preparative silica gelplates (20×20×0.2 cm) in toluene/EtOAc/MeOH (5/4/0.5, v/v/v). Theproduct bands were eluted with CH₂ Cl₂ /MeOH (4:1) and evaporated to asolid foam, which was dried in high vacuum to give 0.064 g (67%) of the5'-hydroxy ApA_(Rp) *ApA 36 and 0.057 g (60%) of the corresponding PStetramer 37. Anal. calc. for 5'-OH-ApA_(Rp) *ApA 36=C₁₂₂ H₁₅₆ N₂₃ O₃₁ P₃SSi₅ ×H₂ O (2724.1): C 53.79, H 5.85, N 11.83; found: C 53.62, H 5.87, N11.51. UV (MeOH): λ_(max) (logε) 277 (5.00); 260 (4.87)!; 92.35 (4.81)!.R_(f) silica gel with toluene/EtOAc/MeOH (5:4:0.5)=0.41. Anal. calc. for5'-OH-ApA_(Sp) *ApA 37=C₁₂₂ H₁₅₆ N₂₃ O₃₁ P₃ SSi₅ ×HO (2724.1): C 53.69,H 5.85, N 11.83; found: C 53.58, H 5,97, N 11.32. UV (MeOH): λ_(max)(logε) 277 (4.96): 260 (4.82)!; 234 (4.74)!. R_(f) silica gel withtoluene/EtOAc/MeOH (5/4/0.5, v/v/v)=0.39.

EXAMPLE 3 a. Adenylyl(2'-5')-(PR)-P-thioadenylyl-(2'-5')-adenylyl-2(2'-5')-adenosine ApA_(Rp)*ApA 38 b. Adenylyl(2'-5')-(PS)-P-thioadenylyl-(2'-5')-adenylyl-2(2'-5')-adenosine ApA_(Sp)*ApA 39

The corresponding 5'-hydroxy tetramers 36 and 37, respectively, wereseparately deblocked by stirring each 5'-hydroxy tetramer (0.056 g;0.021 mmole) with 0.5M DBU in absolute CH₃ CN (2.5 ml) at r.t. and after22 h, the solution was neutralized with 1M AcOH in absolute CH₃ CN (1.25ml) and evaporated to dryness. The residual mixture was then treatedwith methanolic ammonia and after stirring at r.t. for 60 h, the solventwas removed in vacuum and finally the residue was disilylated with 1MBu₄ NF in THF (5 ml) for three days. The solvent was then removed, theresidue was dissolved in HO (10 ml), applied onto a DEAE Sephadex columnA-25 (30×2 cm) and chromatographed first with H₂ O (200 ml) and thenwith a linear gradient of 0-0.04 ml TEAB buffer, pH 7.5, within 3000 ml(flow rate 2 ml/min). Under this condition, the ApA_(Rp) *ApA tetramer38 was eluted with a 0.23-0.28M TEAB buffer and ApA_(Sp) *ApA tetramer39 with 0.245-0.305M TEAB buffer, respectively. The product fractionswere collected, evaporated and coevaporated several times with MeOH. Forfurther purification, paper chromatography was performed using a systemof i-PrOH/ammonia H₂ O (55/10/35, v/v/v). The product band was cut out,eluted with H₂ O, concentrated to a smaller volume and finallylyophilized to give 728 O.D.₂₆₀ nm units (73%) of the ApA_(Rp) *ApAisomer 38 and 686 O.D.₂₆₀ nm units (69%) of the ApA_(Sp) *ApA isomer 39,respectively. ApA_(Rp) *ApA 38: R_(f) on cellulose in i-PrOH/ammonia/H₂O (55:10:35)=0.36. UW (H₂ O): λ_(max) 257 nm. ¹ H-NMR (D₂ O): 8.15,8.07, 8.06, 7.93 (4s, 4H, 4×H--C(8)); 7.92 (s, 2H, 2×H--C(2)); 6.03,5.89, 5.86, 5.79 (4d, 4×H--C(1')). HPLC: on PR-18, A: 50 mM NH₄ H₂ PO₄(pH 7.24). B: MeOH/H₂ O (1/1, v/v); gradient: 0-1 min, 80% A, 20% B;1-31 min, 30% A, 70% B; retention time: 9.55 min. ApA_(Sp) *ApA 39:R_(f) on cellulose in i-PrOH/ammonia/H₂ O (55/10/35, v/v/v)=0.40. UV (H₂O): λ_(max) 257 nm. HPLC: PR-18, A: 50 mM NH₄ H₂ PO₄ (pH 7.24). B:MeOH/H₂ O (1/1, v/v); gradient: 0-1 min, 80% A, 20% B; 1-31 min, 30% A,70% B; retention time: 10.37 min.

Scheme 4 is the beginning of the reaction scheme for the preparation ofthe remaining tetramers from their intermediates, A_(Rp) *Ap-diester 45and the A_(Sp) *Ap-diester 46, with the blocking groups in place. Thepreparation of the diesters is outlined in Preparations 13 and 14.##STR10##

PREPARATION 13 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5')-(monomethoxytrityl)-adenylyl -2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2,5-dichlorophenyl,2-(4-nitrophenyl)-ethylphosphate!ApAp-triester 41 and41a

6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-adenosine-2'-2,5-dichlorophenyl,2-(4-nitrophenyl)ethylphosphate!5'-OH-Ap-triester 40(0.43 g, 0.5 mmole) and the phosphoramidite 15 (0.735 g, 0.7 mmole) weredissolved in absolute CH₃ CN (5 ml) in the presence of tetrazole (0.098g, 1.5 mmole) under a nitrogen atmosphere. After stirring for 3.5 h atr.t., phosphoramidite (0.2 g, 0.19 mmole) and tetrazole (0.026 g, 0.37mmole) were added again and the reaction mixture was stirred for another30 min. A solution of I₂ 0.5 g in Ch₂ Cl₂ /H₂ O/pyridine (1/1/3, v/v/v)!was added dropwise until the brown color did not disappear. The mixturewas stirred for another 10 min, diluted with CH₂ Cl₂ (20 ml) and washedwith saturated Na₂ S₂ O₃ NaCl solution (2×80 ml). The organic phase wascollected, dried over Na₂ SO₄, evaporated and coevaporated with toluene(3×30 ml) to remove the pyridine. The crude product was purified byflash silica gel chromatography (15×2 cm), using toluene/EtOAc (1/1,v/v), EtOAc and EtOAc/2-4% MeOH as eluants. The product fraction wasevaporated to a solid foam, which was dried in high vacuum at 30° C. togive 0.610 g (67%) of the ApAp-triester 41 and 41a. The identity of theisolated compound with authentic material was proved byspectrophotometric comparison. The authentic material was synthesizedfrom the Ap-diester 20 (Charubala et al., 1981) with the 5'-hydroxyP-triester 40 by the phosphotriester method. Anal. calc. forApAp-triester=C₈₈ H₉₄ N₁₂ O₂₀ Cl₂ P₂ SSi₂ (1828.8): C 57.80, H 5.18, N9.9. Found: C 57.77, H 5.20, N 9.02. UV (MeOH): λ_(max) (logε): 277(4.75); 260 (4.62)!; 228 (4.72)!. R_(f) on silica gel withtoluene/EtOAc/MeOH (5/4/1, v/v/v)=0.78.

b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5')-(monomethoxytrityl)-(PR,PS)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2,5-dichlorophenyl-2-(4-nitrophenylethyl)-phosphate!A(_(Rp),Sp)*Ap-triester 43+44

6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-adenosine-2'-2,5-dichlorophenyl,2-(4-nitrohenyl)-ethylphosphate!5'-OH-Ap-triester 40(0.52 g; 0.6 mmole) and the phosphoramidite 15 (0.95 g; 0.9 mmole) weredissolved in absolute CH₃ CN (6.5 ml) in the presence of tetrazole(0.126 g, 1.8 mmole) and under nitrogen atmosphere. After stirring for 3h at r.t., S₈ (0.39 g, 1.51 mmole) and absolute pyridine (3.9 ml) wereadded and the reaction mixture was further stirred for 20 h, thenextracted with CH₂ Cl₂ (2×80 ml) and H₂ O (2×80 ml). The organic phasewas collected, dried over Na₂ SO₄ and evaporated to dryness. Finalcoevaporation was done with toluene (4×20 ml) to remove the pyridine.The crude diastereomeric mixture A(_(Rp),Sp)*Ap-triester 43+44 waspurified by flash silica gel column chromatography (14×2.5 cm), using200 ml CH₂ Cl₂, CH₂ Cl₂ /1% MeOH, 2% MeOH and finally 200 ml CH₂ Cl₂ /1%MeOH, 2% MeOH and finally CH₂ Cl₂ /3% MeOH as eluants. The productfraction (150 ml) was evaporated to a solid foam, which was dried inhigh vacuum to give 0.975 g (88%) of (43) and (44) as a diastereomericmixture. Anal. calc. for A(_(Rp),Sp)*Ap-triester 43+44=C₈₈ H₉₄ N₁₂ O₁₉Cl₂ P₂ SSi₂ (1844.9): C 57.29, H 5.14, N 9.11. Found: C 56.96, H 5.16, N9.09. UV (MeOH): λ_(max) (logε): 277 (4.75); 228 (4.72)!. R_(f) onsilica gel with toluene/EtOAc/CHCl₃ (1/1/1, v/v/v)=0.21. ³¹ P-NMR(CDCl₃) 69.87, 69.25, -6.89, -7.22 and -7.31 ppm.

PREPARATION 14 a. Triethylammonium-N-6-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5')-(monomethoxytrityl)-(PR)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)-ethyl-5'!-N-6-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2-(4-nitrophenylethyl)-phosphate! A_(Rp) *Ap-diester 45 b.Triethylammonium-N-6-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5')-(monomethoxytrityl)-(PS)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)-ethyl -5'!-N-6-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2-(4-nitrophenylethyl)-phosphate! A_(Sp) *Ap-diester 46

The solution of 0.558 g (3.36 mmole) of 4-nitrobenzaldehyde oxime in 15ml of H₂ O/dioxane/Et₃ N (1:1:1) was stirred for 30 min at r.t. Then,0.62 g (0.336 mmole) of the diastereomeric mixture of theA(_(Rp),Sp)*Ap-triester 43+44 was added and stirred for 2.5 h at r.t.The mixture was evaporated, then coevaporated with pyridine (3×15 ml),toluene (3×15 ml) and finally with CH₂ Cl₂ (3×15 ml). The residue wasdissolved in a small amount of CHCl₃ and chromatographed on a flashsilica gel column (15×2.5 cm) with CHCl₃ (150 ml), CHCl₃ /2% MeOH (200ml), 4% MeOH (100 ml), 6% MeOH (200 ml), CHCl₃ /6% MeOH/0.5% Et₃ N (300ml) and CHCl₃ /6% MeOH/2% Et₃ N (250 ml). The product fraction (600 ml)was evaporated to a solid foam, which was dried under high vacuum togive 0.55 g (91%) of the isomeric mixture 45+46. Separation into thepure diastereomers was achieved by chromatography on preparative silicagel plates (7 plates, 40×20×0.2 cm) and three developments in CHCl₃/MeOH (9/1, v/v). The product bands were eluted with CHCl₃ /MeOH (4/1,v/v) containing 1% Et₃ N and evaporated to a solid foam to give 0.262 g(43%) of A_(Rp) *Ap-diester 45, 0.144 (24%) of A_(Sp) *Ap-diester 46 and0.045 g (7%) of A(_(Rp),Sp)*Ap-diester 45+46. Anal. calc. for A_(Rp)*Ap-diester 45=C₈₈ H₁₀₇ N₁₃ O₁₉ P₂ SSi₂ ×2 H₂ O (183.7.1) C 57.53, H6.09, N 9.91. Found: C 57.30, H 6.70, N 9.75. UV (MeOH): λ_(max) (logε);277 (4.69); 260 (4.56)!; 231 (4.61)!. R_(f) on silica gel with CHCl₃/MeOH (9/1, v/v)=0.37. ³¹ P-NMR (CDCl₃): 69.66 and -0.09 ppm. Anal.calc. for A_(Sp) *Ap-diester 46=C₈₈ H₁₀₇ N₁₃ O₁₉ P₂ SSi₂×2 H₂ O(1837.1): C 57.53, H 6.09, N 9.91. Found: C 56.44, H 7.15, N 8.61. UV(MeOH): λ_(max) (logε); 276 (4.60); 260 (4.49)!; 232 (4.52)!. R_(f) onsilica gel with CHCl₃ /MeOH (9/1, i/v)=0.28. ³¹ P-NMR (CDCl₃): 68.96 and-0.06 ppm.

Scheme 5 is the reaction scheme for the preparation of the remainingfully resolved tetramers, ApApA_(Rp) *A 51, ApApA_(Sp) *A 52, A_(Rp)*ApApA 57, and A_(Sp) *ApApA 58, from their respective protectedintermediates, 47, 48, 53 and 54. The corresponding preparations areoutlined in Preparation 15, 16, and 17 and Examples 4 and 5. ##STR11##

PREPARATION 15 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)-dimethylsilyl!-adenosine ApApA_(Rp) *A 47

Triethylammonium 6-N-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2-(4-nitrophenyl)ethyl-phosphate! 42 (0.14 g; 0.078 mmole) and the5'-hydroxy PR dimer 18 (0.08 g, 0.06 mole) were coevaporated with drypyridine (4×0.5 ml), dissolved in dry pyridine (0.6 ml) and then(2,4,6-triisopropyl)benzenesulfonyl chloride (0.047 mg, 0.156 mmole) and3-nitro-1,2,4-triazole (0.053 mg, 0.47 mmole) were added. The solutionwas stirred at r.t. for 22 h, extracted with CH₂ Cl₂ (2×30 ml), washedwith H₂ O (2×20 ml), dried over Na₂ SO₄ and evaporated to dryness.Pyridine was removed by coevaporation with toluene (3×20 ml). The crudetetramer 47 was purified by flash silica gel column chromatography (15×1cm) and eluted first with CH₂ Cl₂ (50 ml), then with CH₂ Cl₂ /1% MeOH(100 ml), 2% MeOH (50 ml) and finally with CH₂ Cl₂ /3% MeOH (100 ml).The product fraction (80 ml) was evaporated to dryness to give 0.11 g(62%) of the fully protected tetramer ApApA_(Rp) *A 47 as a colorlessfoam after drying under high vacuum at 350° C. Anal. calc. for C₁₄₂ H₁₇₂N₂₃ O₃₂ P₃ SSi₅ ×H₂ O (2996.5): C 56.92, H 5.85, N 10.75. Found: C56.51, H 5.91, N 10.37. UV (MeOH): λ_(max) (logε) 277 (4.99); 259(4.84)!; 233 (4.85)!. R_(f) on silica gel with CHCl₃ /MeOH (19/1,v/v)=0.46.

b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)-dimethylsilyl!-adenosine ApApA_(Sp) *A 48

ApAp-diester 41 (0.14 g, 0.078 mmole) and the 5'-hydroxy PS dimer 19(0.08 g, 0.06 mmole) were coevaporated with dry pyridine (4×0.5 ml),dissolved in dry pyridine (0.6 ml) and then(2,4,6-triisopropyl)benzenesulfonyl chloride (0.47 mg, 0.156 mmole) and3-nitro-1,2,4-triazole (0.053 mg, 0.46 mmole) were added. After stirringfor 4.5 h at r.t., (2,4,6-triisopropyl)benzenesulfonyl chloride (0.024g, 0.078 mmole) and 3-nitro-1,2,4-triazole (0.027 g, 0.234 mmole) wereadded again. The solution was stirred at r.t. for 16.5 h, then extractedwith CH₂ Cl₂ (4×20 ml) and added with H₂ O (3×20 ml), dried over Na₂ SO₄and evaporated. Final coevaporations were done with toluene (4×15 ml) toremove pyridine. The crude tetramer 48 was purified by flash silica gelcolumn chromatography (15×1 cm) and eluted analogous to tetramer 47 withCH₂ Cl₂ and CH₂ Cl₂ /1-3% MeOH to give 0.107 g (60%) of the fullyprotected tetramer ApApA_(Sp) *A 48 as a colorless foam after dryingunder high vacuum at 35° C. Anal. calc. for C₁₄₂ H₁₇₂ N₂₃ O₃₂ P₃ SSi₅×H₂ O (2996.5): C 56.92, H 5.85, N 10.75. Found: C 56.51, H 5.91, N10.85. UV (MeOH): λ_(max) (logε) 277 (4.99); 259 (4.85)!; 231 (4.86)!.R_(f) on silica gel with CHCl₃ /MeOH (19/1, v/v)=0.46.

PREPARATION 16 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5'-OH-ApApA_(Rp) *A 49 b.6-N-Benzoyl-3'-O- (tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5'-OH-ApApA_(Sp) *A 50

The fully protected tetramer ApApA_(Rp) *A 47 (0.104 g, 0.035 mmole) wasstirred with 2% p-TsOH in CH₂ Cl₂ /MeOH (4/1, v/v, 1.4 ml) at r.t. After1.5 h, the reaction mixture was extracted with CH₂ Cl₂ (3×40 ml) and H₂O (2×40 ml). The combined organic phase was dried over Na₂ SO₄ andevaporated to dryness. The crude product was purified on a flash silicagel column (11×1 cm) and the product eluted with 20 ml CH₂ Cl₂ and 50 mlCH₂ Cl₂ /1% MeOH to 5% MeOH. The product fraction (100 ml) wasevaporated and dried under high vacuum to give 0.075 g (80%) of thehydroxy tetramer ApApA_(Rp) *A 49. Anal. calc. for C₁₂₂ H₁₅₆ N₂₃ O₃₁ P₃SSi₅ ×H₂ O (2706.5): C 54.15, H 5.81, N 11.90. Found: C 53.97, H 6.02, N11.65. UV (MeOH): λ_(max) (logε) 277 (5.00); 260 (4.85)!; 234 (4.77)!.R_(f) on silica gel with CHCl₃ /MeOH (19/1, v/v)=0.43.

The fully protected tetramer ApApA_(Sp) *A 48 was treated in ananalogous manner through the purification stage. The crude product 50was purified on two preparative silica gel plates (20×20×0.2 cm) inCHCl₃ /MeOH (19/1, v/v), the product band was eluted with CH₂ Cl₂ /MeOH(4/1, v/v) and evaporated to a solid foam to give 0.068 g (72%) of the5'-hydroxy tetramer ApApA_(Sp) *A 50.

EXAMPLE 4 a.Adenylyl-(2'-5')-adenylyl-(2'-5')-(PR)-P-thioadenylyl-(2'-5')-adenosineApApA_(Rp) *A 51 b.Adenylyl-(2'-5')-adenylyl-(2'-5')-(PS)-P-thioadenylyl-(2'-5')-adenosineApApA_(Sp) *A 52

The corresponding 5'-hydroxy tetramers 49 and 50, respectively, weredeblocked separately by stirring the 5'-hydroxy tetramer (0.067 g, 0.025mmole) with 0.5M DBU in absolute CH₃ CN (3 ml) at r.t. for 20 h, thesolution was neutralized with 1M AcOH in absolute CH₃ CN (1.5 ml) andevaporated to dryness. R_(f) on silica gel with {EtOAc/i-PrOH/ammonia/H₂O, 7/1/2, v/v/v) 7/3, v/v: ApApA_(Rp) *A=0.58; ApApA_(Sp) *A=0.66. Theresidue was then treated with methanolic ammonia (10 ml) and after 3days reaction time, the solvent was removed under vacuum. R_(f) onsilica gel with EtOAc/i-PrOH/ammonia/H₂ O, 7/1/2, v/v/v) 1/1, v/v}:ApApA_(Rp) *A=0.38; ApApA_(Sp) *A=0.36!. Desilylation was done with 1MBu₄ NF in THF (5 ml). The reaction mixture was stirred at r.t. for 48 hand then the solvent was evaporated in vacuum. The residue was taken upin H₂ O (10 ml) and applied to a DEAE Sephadex A-25 column (30×2 cm).With flow rates of 2 ml/min, the pure tetramer ApApA_(Rp) *A was elutedwith 0.15-0.20M TEAB buffer, pH 7.5, and in the case of the tetramerApApA_(Sp) *A with 0.24-0.32M TEAB buffer, pH 7.5. After evaporation andcoevaporation with MeOH several times, the tetramer was applied ontoeight paper sheets (25×50 cm) and developed in i-PrOH/ammonia/H₂ O(6/1/3, v/v/v). The product band was cut out, eluted with H₂ O,concentrated to a smaller volume and finally lyophilized to give 675O.D.₂₆₀ nm units (57%) of ApApA_(Rp) *A isomer 51 and 753 O.D.₂₆₀ nm(65%) of ApApA_(Sp) *A isomer 52. ApApA_(Rp) *A 51: R_(f) on cellulosein i-PrOH/ammonia/H₂ O (6/1/3, v/v/v)=0.33. UV (H₂ O): λ_(max) 258 nm.HPLC: PR-18, A: 50 mM NH₄ H₂ PO₄, pH 7.2. B: MeOH/H₂ O (1/1, v/v),gradient: 0-1 min, 80% A, 20% B; 1-31 min, 30% A, 70% B; retention time:9.70 min. ApApA_(Sp) *A 52: R_(f) on cellulose in i-PrOH/ammonia/H₂ O(6/1/3, v/v/v)=0.21. UV (H₂ O): λ_(max) 258 nm. HPLC: PR-18, A: 50 mMNH₄ H₂ PO₄, pH 7.2. B: MeOH/H₂ O (1/1, v/v), gradient: 0-1 min, 80% A,20% B; 1-31 min, 30% A, 70% B; retention time: 13.49 min.

PREPARATION 17 a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A_(Rp) *ApApA 53

Triethylammonium 6-N-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'- (O^(P)-2-(4-nitrophenyl)ethyl-phosphate! A_(Rp) *Ap-diester 45 (0.141 g, 0.078mmole) and the 5'-hydroxy dimer 29 (0.078 g, 0.06 mmole) werecoevaporated with dry pyridine (4×0.5 ml) and finally dissolved in drypyridine (0.6 ml). Then (2,4,6-triisopropyl)-benzenesulfonyl chloride(0.047 mg, 0.156 mmole) and 3-nitro-1,2,4-triazole (0.053 mg, 0.47mmole) were added and stirred at r.t. for 21 h. The reaction mixture wasdiluted with CH₂ Cl₂ (60 ml) and washed with H₂ O (2×30 ml), dried overNa₂ O₄ and evaporated to dryness. Pyridine was removed by coevaporationwith toluene (3×20 ml). The crude tetramer 53 was purified by flashsilica gel column chromatograpy (11×1 cm) and eluted first with CH₂ Cl₂(50 ml), then with CH₂ Cl₂ /1% MeOH (100 ml), 2% MeOH (200 ml), 3% MeOH(50 ml) and finally with CH₂ Cl₂ /5% MeOH (50 ml). The product fraction(200 ml) was evaporated to dryness. The residue was chromatographedagain on two preparative silica gel plates (20×20×0.2 cm) intoluene/EtOAc/MeOH (5/4/0.5, v/v/v) to remove small amount of 5'-hydroxydimer. The tetramer product band was eluted with CH₂ Cl₂ /MeOH (4/1,v/v) and evaporated to a solid foam to give 0.053 g (30%) of thetetramer A_(Rp) *ApApA 53 after drying in high vacuum at 35° C. Anal.calc. for C₁₄₂ H₁₇₂ N₂₃ O₃₂ P₃ SSi₅ ×3 H₂ O (3032.5): C 56.24, H 5.92, N10.62. Found: C 55.75, H. 5.71, N 9.83. UV (MeOH): λ_(max) (logε) 277(4.96); 260 (4.82)!; 232 (4.82)!. R_(f) on silica gel withtoluene/EtOAc/MeOH (5:4:1)=0.78.

b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PS)-P-thioadenylyl-2'-(O^(P) -2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'- (O^(P)-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine A_(Sp) *ApApA 54

A_(Sp) *Ap-diester 46 (0.141 g, 0.078 mmole) and the 5'-hydroxy dimer 29(0.078 g, 0.06 mmole) were coevaporated with dry pyridine (4×0.5 ml) anddissolved in dry pyridine (0.6 ml). (2,4,6-Triisopropyl)-benzenesulfonylchloride (0.047 mg, 0.156 mmole) and 3-nitro-1,2,4-triazole (0.053 mg,0.47 mmole) were added and the mixture was stirred at r.t. After 21 h,(2,4,6-triisopropyl)benzenesulfonyl chloride (0.024 mg, 0.079 mmole) and3-nitro-1,2,4-triazole (0.027 mg, 0.24 mmole) were added again. Thereaction mixture was stirred for another hour, then diluted with CH₂ Cl₂(60 ml) and washed with H₂ O (2×30 ml), dried over Na₂ SO₄ andevaporated to dryness. Further work-up was performed analagous to thatdescribed for tetramer 53 to give 43 mg (24%) of A_(Sp) *ApApA 54 in theform of a solid foam. Anal. calc. for C₁₄₂ H₁₇₂ N₂₃ O₃₂ P₃ SSi₅ ×H₂ O(2996.5): C 56.92, H 5.85, N 10.75. Found: C 56.63, H 6.08, N 10.18. UV(MeOH): λ_(max) (logε) 277 (4.98); 260 (4.84)!; 232 (4.85)!. R_(f) onsilica gel with toluene/EtOAc/MeOH (5/4/1, v/v/v)=0.78.

EXAMPLE 5 a.(PR)-P-Thioadenylyl-(2'-5')-adenylyl-(2'-5')-adenylyl-(2'-5')-adenosineA_(Rp) *ApApA 57 b.(PS)-P-Thioadenylyl-(2'-5')-adenylyl-(2'-5')-adenylyl-(2'-5')-adenosineA_(Sp) *ApApA 58

The corresponding fully protected tetramers 53 and 54 were deblocked bystirring a solution of 0.047 g (0.016 mmole) of PR tetramer 53 (PStetramer 54: 0.032 g, 0.012 mmole) in 2% p-TsOH in CH₂ Cl₂ /MeOH (4/1;v/v; for PR: 0.5 ml; for PS: 0.38 ml) for 1 h at r.t. The reactionmixture was diluted with CH₂ Cl₂ (60 ml), washed with H₂ O (2×30 ml),dried over Na₂ SO₄ and evaporated to dryness. The crude products werepurified on preparative silica gel plates (20×20×0.2 cm) in CHCl₃ /MeOH(19/1, v/v), the product bands were eluted with CH₂ Cl₂ /MeOH (4/1, v/v)and evaporated to solid foams to give 0.034 g (80%) of the 5'-hydroxyA_(Rp) *ApApA isomer 55 and 0.02 g (68%) of the 5'-hydroxy A_(Rp) *ApApAisomer 56. The solution of the 5'-hydroxy tetramers 55 (0.034 g, 0.013mmole) and 56 (0.02 g, 0.007 mmole), respectively, were separatelystirred with 0.5M DBU in absolute CH₃ CN (55: 1.5 ml; 56: 0.9 ml)! for18 h at r.t., then neutralized by addition of 1M AcOH (55: 0.75 ml; 56:0.45 ml)! and evaporated. The residue was treated with 10 ml ofsaturated methanolic ammonia and the solution, after stirring at r.t.for 60 h, was evaporated to dryness. Desilylation was done by treatmentwith 1M Bu₄ NF in THF (2.5 ml). After stirring at r.t. for 60 h, thesolvent was removed under vacuum. Some H₂ O (10 ml) was added to theresulting residue and applied to a DEAE Sephadex column A-25 (32×2 cm)and eluted with 0-0.5M TEAB buffer, pH 7.5. The fractions of the mainpeak were collected, evaporated and coevaporated several times withMeOH. Further purification by paper chromatography (i-PrOH/ammonia/H₂ O,55/10/35, v/v/v) gave, after lyophilization, 347 O.D.₂₆₀ nm units (58%)of A_(Rp) *ApApA 57 and 111 O.D.₂₆₀ nm units (31%) of A_(Sp) *ApApA 58,respectively. A_(Rp) *ApApA 57: UV (H₂ O)=257 nm. R_(f) on cellulose ini-PrOH/ammonia/H₂ O (6/1/3, v/v/v)=0.21. HPLC: PR-18, A: 50 mM NH₄ H₂PO₄ (pH 7.2). B: MeOH/H₂ O (1/1, v/v), gradient: 0-1 min, 80% A, 20% B;1-31 min, 30% A, 70% B; retention time: 7.47 min. A_(Sp) *pApA 58: UV(H₂ O)=257 nm. R_(f) on cellulose in iPrOH/ammonia/H₂ O (6/1/3,v/v/v)=0.32. HPLC: PR-18, A: 50 mM NH₄ H₂ PO₄, pH 7.2. B: MeOH/H₂ O(1/1, v/v), gradient: 0-1 min, 80% A, 20% B; 1-31 min, 30% A, 70% B;retention time: 9.84 min.

Preparation of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylate5'-Monophosphates

The phosphorothioate/phosphodiester trimer and tetramer cores weresynthesized as described above in Examples 1, 2, 3, 4, and 5. The trimerand tetramer 5'-monophosphates were enzymatically synthesized accordingto the procedure of Sambrook et al., Molecular Cloning--A LaboratoryManual, 2 ed., Cold Spring Harbor Laboratory Press, pp. 5.68-5.71 (1989)from their corresponding cores and ATP with T4 polynucleotide kinase.5'-Monophosphorylation was determined by reverse-phase HPLC analysis andconfirmed by the subsequent hydrolysis of each 5'-monophosphatederivative by 5'-nucleotidase (data not shown). Yields ofphosphorylation ranged from 15% to 68%.

Preparation of 2',5'-Phosphorothioate/phosphodiester Oligoadenylate5'-Diphosphate and 5'-Triphosphate

The 5'-diphosphate and 5-triphosphate of the2',5'-phosphorothioate/phosphodiester oligoadenylates may be preparedfrom the 5'-monophosphate by following the procedure of Example 6.

EXAMPLE 6

All reactions are performed in glassware oven-dried at 125° C. for 18-24hr. A 2',5'-phosphorothioate/phosphodiester oligoadenylate stereoisomer(trimer or tetramer, 400 OD units at 260 nm) is dissolved in 500microliters of dry dimethylformamide ("DMF") and dried in vacuo in a 10ml conical flask at 35° C. This process is repeated three times. To thedry residue, 50 micromoles of triphenylphosphine, 100 micromoles ofimidazole and 50 micromoles of dipyridinyl disulfide are added. Themixture is dissolved in 500 microliters dry DMF plus 50 microliters ofdry dimethylsulfoxide. The solution is stirred with a stirring bar for 2hr at room temperature. After 2 hr the solution is homogeneous (after 30minutes, the solution begins to change to yellow). The solution istransferred dropwise to 10 ml of a 1% NaI/dry acetone (w/v) solution.The clear colorless precipitate which forms is the sodium salt of the5'-phosphoroimidazolidate. The precipitate is centrifuged at roomtemperature, the supernatant is decanted, and the precipitate is washedthree times with 10 ml dry acetone. The centrifuging is repeated. Theprecitipate is dried over P₂ O₅ in vacuo for 2 hr. The precipitate isdissolved in 200 microliters of freshly prepared 0.5M tributylammoniumpyrophosphate in dry DMF. The solution is maintained at room temperaturefor 18 hr after which time the DMF is removed in vacuo. The residue isdissolved in 0.25M triethylammonium bicarbonate buffer ("TEAB") (pH7.5). The 5'-di and 5'-triphosphate products are separated using aDEAE-Sephadex A25 column (HCO₃ -form; 1×20 cm) with a linear gradient of0.25M to 0.75N TEAB. Fractions (10 ml) are collected. The product isobserved by ultraviolet spectroscopy at 254 nm. The fractions containingthe 5'-di and 5'-triphosphates are separately pooled and dried in vacuo.by lyophilization. The yield of the 5'-diphosphate is about 5%; theyield of the 5'-triphosphate is about 60%.

Stability of the Phosphorothioate/Phosphodiester Trimer and TetramerCore Derivatives to Serum Phosphodiesterase

The stability of authentic 2-5A and phosphorothioate/phosphodiestertrimer and tetramer core derivatives (300 μM) was determined byincubation in 200 μL of RPMI-1640 medium supplemented with 10% fetalcalf serum in 5% CO₂ -in-air at 37° C. Aliquots (30 μL) were removed attime zero and 6 hours. The hydrolysis products were identified by HPLCas described in Kariko et al. , Biochemistry 26: 7127-7135 (1987). Underthe conditions described therein, authentic A₂ and A₃ were completelyhydrolyzed to inosine and hypoxanthine in 20 min (Table 1), while A_(Rp)*A and A_(Sp) *A were not hydrolyzed.

No hydrolysis of the phosphorothioate/phosphodiester trimer corederivatives was observed. However, the phosphorothioate/phosphodiestertetramer core derivatives were hydrolyzed from the 5'- or2',3'-terminus, depending on the location of thephosphorothioate-substituted internucleotide linkage. For example,A_(Rp) *ApApA and A_(Sp) *ApApA were 50% degraded to their respectivedimer cores, A_(Rp) *A and A_(Sp) *A, whereas ApA_(Rp) *ApA and ApA_(Sp)*ApA are degraded from the 2',3'-terminus to form the trimer cores,ApA_(Rp) *A and ApA_(Sp) *A. ApApA_(Rp) *A and ApApA_(Sp) *A aredegraded from the 5'-terminus to yield ApA_(Rp) *A and ApA_(Sp) *A,respectively.

                  TABLE 1    ______________________________________    Hydrolysis of Phosphorothioate/Phosphodiester Trimer and Tetramer    2-5A Core Derivatives by Serum Phosphodiesterases    2-5A or    Derivative              % Hydrolysis.sup.a                             Hydrolysis Products.sup.b    ______________________________________    A.sub.2   100 (20 min)   inosine, hypoxanthine    A.sub.3   100 (20 min)   inosine, hypoxanthine    A.sub.Rp *A               0.sup.b       not hydrolyzed    A.sub.Sp *A               0.sup.b       not hydrolyzed    A.sub.Rp *ApA              0              not hydrolyzed    A.sub.Sp *ApA              0              not hydrolyzed    ApA.sub.Rp *A              0              not hydrolyzed    ApA.sub.Sp *A              0              not hydrolyzed    A.sub.Rp *ApApA              100            inosine, hypoxanthine,                             A.sub.Rp *A    A.sub.Sp *ApApA              100            inosine, hypoxanthine,                             A.sub.Sp *A    ApA.sub.Rp *ApA              50             inosine, hypoxanthine,                             A.sub.Rp *ApA    ApA.sub.Sp *ApA              50             inosine, hypoxanthine,                             A.sub.Sp *ApA    ApApA.sub.Rp *A              30             inosine, hypoxanthine,                             ApA.sub.Rp *A    ApApA.sub.Sp *A              33             inosine, hypoxanthine,                             ApA.sub.Sp *A    ______________________________________     .sup.a Incubations were for 6 h as described in text. Number in     parentheses indicates the time at which 100% hydrolysis was observed.     .sup.b Identified as described by Kariko et al., Biochemistry 26: 7127713     (1987).

Binding of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates toRNase L

The affinity of the phosphorothioate/phosphodiester 2-5A derivatives forRNase L was determined in radiobinding assays according to the method ofKnight et al., Meth. Enzymol 79: 216-227 (1981). Authentic A₃, pA₃ andp₃ A₃ bind to RNase L with IC₅₀, values of 1×10⁻⁶ M, 1×10⁻⁹ M and 1×10⁻⁹M, respectively. According to the invention, the binding of thephosphorothioate/phosphodiester core and their 5'1-monophosphates toRNase L was equivalent to or slightly better than the correspondingauthentic 2-5A cores and 5'-monophosphates, with IC₅₀ values from 8×10⁻⁷M to 8×10⁻⁶ M for the cores and from 1×10⁻⁸ M to 1×10⁻⁹ M for the5'-monophosphates. The trimer and tetramer 2-5A core derivatives withphosphorothioate substitution in the first internucleotide linkage fromthe 5'-terminus exhibited lower affinity compared to those withphosphorothioate substitution in the second or third internucleotidelinkage.

Activation of RNase L by Phosphorothioate/Phosphodiester Derivatives of2-5A

Correlation of biological properties with absolute configuration hasonly been possible with the preparation of the fully resolved2',5'-phosphorothioate/phosphodiester adenylate trimer cores. However,the trimer core compounds have been found to bind and/or activate RNaseL only modestly. RNase L activation by the 2',5'-phosphorothioate coremolecules is significantly enhanced by 5'-phosphorylation.

Core-cellulose assays were performed according to the method ofSilverman, Anal. Biochem. 144: 450-460 (1985) and Kariko et al. (1987),supra, in which RNase L was partially purified from L929 cell extractsby immobilization on 2-5A₄ core-cellulose. Activation of RNase L wasmeasured by the conversion of poly(U) ³² p!pCp to acid solublefragments. The results indicate that authentic p₃ A₃, p₃ A₄, pA₃ and pA₄have IC₅₀ values of 5×10⁻¹⁰ M, 5×10⁻¹⁰ M, 2×10⁻⁷ M and 2×10⁻⁸ M,respectively, while surprisingly, of the phosphorothioate/phosphodiestertrimer core derivatives, only ApA_(Rp) *A can activate RNase L (IC₅₀ of5×10⁻⁷ M) (FIG. 1A, Δ). Three of the phosphorothioate/phosphodiestertrimer 5'-monophosphates can activate RNase L, with pApA_(Rp) *A beingthe most potent activator of RNase L (IC₅₀ of 1×10⁻⁹ M) (FIG. 1B, Δ).pA_(Rp) *ApA (□) and pA_(Sp) *ApA (▪) are 100-fold less potentactivators of RNase L. Of the six phosphorothioate/phosphodiestertetramer core derivatives, only ApA_(Rp) *ApA (□) and ApApA_(Rp) *A (Δ)can activate RNase L (IC₅₀ of 5×10⁻⁷ M and 5×10⁻⁷ M, respectively) (FIG.1C). Five of the six phosphorothioate/phosphodiester tetramer5'-monophosphates activate RNase L (IC₅₀ values >6×10⁻⁷ M to 8×10⁻¹⁰ M).The pApA_(Sp) *ApA enantiomer did not activate RNase L, even atconcentrations as high as 1×10⁻⁵ M (FIG. 1D, ▪).

Activation of RNase L by the phosphorothioate/phosphodiester trimer andtetramer 2-5A derivatives was also measured in a rRNA cleavage assayusing L929 cell extracts according to the method of Kariko et al.(1987), supra, in which extracts of L929 cells were incubated for 1 h at30° C. in the presence or absence of 2-5A or 2-5A derivative. Consistentwith the results from the core-cellulose assays (FIG. 1A), ApA_(Rp) *A(1×10⁻⁶ M) was the only trimer core able to activate RNase L to cleaverRNA to the well-characterized specific cleavage products (SCP) of RNaseL (FIG. 2A, lane 5). A_(Rp) *ApA, A_(Sp) *ApA and ApA_(Sp) *A, as wellas authentic A₃, did not activate RNase L at concentrations as high as1×10⁻⁶ M (FIG. 2A, lanes 3, 4, 6, 7). Authentic p₃ A₃ was active at1×10⁻⁸ M (FIG. 2A, lane 2). The corresponding 5'-monophosphates, pA_(Rp)*ApA, pA_(Sp) *ApA and pApA_(Rp) *A, activated at 1×10⁻⁷ M, 1×10⁻⁷ M,and 2×10⁻⁹ M, respectively (FIG. 2B, lanes 4-6), as compared with pA₃which was active at 1×10⁻⁶ M (lane 3). Incubation with pApA_(Sp) *A,even at concentrations as high as 5×10⁻⁶ M, did not result in detectablerRNA degradation (data not shown).

Comparable degradation of rRNA was observed with two of the sixphosphorothioate/phosphodiester tetramer core derivatives relative tothe authentic p₃ A₄ control. ApA_(Rp) *ApA and ApApA_(Rp) *A activatedRNase L at 1×10⁻⁵ M (FIG. 3A, lanes 6 and 8). As was observed in thecore-cellulose assays (FIG. 1D), five of the sixphosphorothioate/phosphodiester tetramer 5'-monophosphates were able toactivate RNase L (pA_(Rp) *ApApA, pA_(Sp) *ApApA, pApA_(Rp) *ApA,pApApA_(Rp) *A, and pApApA_(Sp) *A) (FIG. 3B, lanes 4, 5, 6, 8, 9,respectively). The most efficient activator or RNase L was pApA_(Rp)*ApA (1×10⁻⁸ M) (FIG. 3B, lane 6), while pApA_(Sp) *ApA was anantagonist of RNase L activation and was unable to activate RNase L evenat concentrations as high as 1×10⁻⁵ M (FIG. 3B, lane 7).

Inhibition of RNase L Activation by pApA_(Sp) *A

The high affinity of pApA_(Sp) *A for RNase L and the observation thatpApA_(Sp) *A does not activate RNase L, suggests that it might be aspecific inhibitor of RNase L. Indeed, pApA_(Sp) *A inhibits theactivation of RNase L by p₃ A₃ or pApA_(Rp) *A (FIG. 4A). Authentic p₃A₃ activates RNase L to hydrolyze 28S and 18S rRNA to SCP at 10⁻⁹ M or10⁻⁸ M (lanes 1 and 3). However, addition of pApA_(Sp) *A (10⁻⁶ M )results in the inhibition of RNase L-catalyzed hydrolysis of rRNA (lanes2 and 4). Similarly, whereas pApA_(Rp) *A activates RNase L at 10⁻⁹ M or10⁻⁸ M (lanes 5 and 7), the addition of pApA_(Sp) *A (10⁻⁶ M ) inhibitsthis activation (lanes 6 and 8). The inhibitory activity of pApA_(Sp) *Awas also observed with partially-purified RNase L (FIG. 4B). p₃ A₃activates RNase L with an IC₅₀ value of 5×10⁻¹⁰ M (); however, uponaddition of pApA_(Sp) *A (1×10⁻⁶ M ), the observed IC₅₀ value shifts to1×10⁻⁸ M (∘), demonstrating specific inhibition of p₃ A₃ -mediatedactivation of RNase L by pApA_(Sp) *A.

Notwithstanding, pApA_(Sp) *A is useful as a probe in the evaluation ofthe role of RNase L in the interferon-induced biological cascade. Mostimportantly, pApA_(Sp) *A selectively inhibits activation of RNase L atphysiological concentrations, and is metabolically stable to specificand non-specific phosphodiesterases. The molecule provides the means toselectively inhibit RNase L activation.

Moreover, it is expected that pApA_(Sp) *A has therapeutic activity.Individuals afflicted with chronic myelogenous leukemia ("CML") displaya highly elevated RNase L activity, as evidenced by novel rRNACML-specific cleavage products. Thus, pApA_(Sp) *A, which is ametabolically stable inhibitor of RNase L, has potential utility intreating chronic myelogenous leukemia.

Additionally, individuals afflicted with chronic fatigue syndrome("CFS") (also known as myalgic encephalomyelitis (ME) or low naturalkiller ("NK") cell disease) and other HHV-6 related disorders alsodisplay a highly elevated RNase L activity compared to controls (meanbasal level=466±23 compared to 123±12 in controls; p<0.0001), Suhadolniket al, Clinical Infectious Disease 18 (SUPPL. 1): 96-104 (1994). Inexperiments performed using extracts of peripheral blood mononuclearcells ("PBMC") from individuals with CFS before and during therapy witha biological response modifier, poly (I)-poly (C₁₂ U) (mismatched dsRNS,Ampligen®), as compared to healthy individuals, the mean basal latent2-5A synthetase level in PMBC extracts was significantly decreasedfollowing therapy (610±220 picomoles 2-5A/mg protein/hour) compared tocontrols (2035±325 picomoles 2-5A/mg protein/hour, P<0.001). Id.Further, all pretherapy PBMC extracts tested were positive for humanherpes virus-6 (HHV-6) replication. Therapy resulted in a significantdecrease in HHV-6 activity (p<0.01) and down regulation of the 2-5Asynthetase/RNase L pathway in temporal association with clinical andneuropsychological improvement. Without wishing to be bound by anytheory, it appears that the upregulated 2-5A pathway observed in CFSpretherapy is consistent with a hypothesis that an activated immunestate and persistent viral infection may play a pathogenesis of CFS.Thus, pApA_(Sp) *A, which, as stated above, is a metabolically stableinhibitor of RNase L, also has potential utility in treating CFS.

Inhibition of Cell Growth by Phosphorothioate/Phosphodiester 2-5A CoreDerivatives

Cell viability was determined by Trypan blue exclusion. Post-treatmentcolony forming ability was determined by growth in microtiter wells asoutlined in the procedure of Kraemer et al. Mutation Res. 72: 285-292(1980). The results indicate that no decrease in survival or inhibitionof Sup T1 cell growth in microtiter plates was observed with any of thedimer, trimer or tetramer phosphorothioate/phosphodiester 2-5A corederivatives. On the basis of the lack of cytotoxicity and estimateduptake of 1% on a previous report with the cordycepin derivative of2-5A, Suhadolnik et al., Nucleosides and Nucleotides 2: 351-366 (1983),3×10⁻⁴ M was chosen as the concentration at which to screen thephosphorothioate/phosphodiester derivatives for anti-HIV-1 activity.

Effect of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates onInhibition of HIV-1-Induced Syncytia Formation

The infected centers assay as described by Henderson et al., Virology182: 186-198 (1991), was used to measure the ability of thephosphorothioate/phosphodiester derivatives of 2-5A trimer and tetramercores to inhibit HIV-1 induced syncytia formation, an indicator of HIV-1replication in T cells. Freshly isolated peripheral blood lymphocytes(PBL) were treated with 2-5A or derivatives for 2 h and infected withHIV-1 strain IIIB at a m.o.i. of approximately 0.1. The infected PELwere maintained in RPMI-1640 medium supplemented with 10% (v/v)heat-inactivated fetal calf serum at 37° C. in a humidified 5% CO₂ inair atmosphere. After 48 h, the cells were washed twice in Hank'sbalaned salt solution, serially diluted and seeded into multiple wellsof a 96-well microtiter plate. Immediately, 2×10⁵ exponentially growingSup T1 cells were added to each well; Sup T1 cells readily form asyncytium with a cell which is productively infected with HIV-1. Thewells were examined daily for the presence of syncytia, using a tissueculture microscope. The first signs of syncytia formation can be seen in12 h, with some complete syncytia developing by 24 h. Final results wereread at 72 h. Each syncytium was counted as a single infected cell. Thenumber of syncytia per seeded cell is determined and expressed as aninfected center per infected cell. In the control (no 2-5A derivativeadded), 100% syncytia formation was equivalent to 12±3 syncytia per 200HIV-1 infected cells.

The data is shown in FIGS. 5A and 5B. As shown in FIG. 5A, ApA_(Rp) *Awas a highly efficient inhibitor of syncytia formation, with 100%inhibition observed at 3×10⁻⁴ M. Its PS enantiomer, ApA_(Sp) *Ainhibited syncytia formation 78%. A_(Rp) *ApA and A_(Sp) *ApA inhibitedsyncytia formation only 10% and 15%, respectively. Authentic A₃ and A₄(3×10⁻⁴ M) inhibited syncytia formation 21% and 15%, respectively whileadenosine (9×10⁻⁴ M) did not inhibit syncytia formation. Of the sixphosphorothioate/phosphodiester tetramer core derivatives, ApApA_(Rp) *Aand ApApA_(Sp) *A were the most inhibitory (90% and 76% inhibition,respectively) (FIG. 5B). ApA_(Rp) *ApA, ApA_(Sp) *ApA, A_(Rp) *ApApA andA_(Sp) *ApApA inhibited syncytia formation 26%, 32%, 16% and 18%,respectively. Adenosine and A₄ (FIG. 5B), as well as the A_(Rp) *A andA_(Sp) *A dimers, adenine or 3',5'-A₄ (data not shown), were not able toinhibit syncytia formation.

Effect of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates on HIV-1Reverse Transcriptase Activity

Sup T1 cells were treated with 2-5A or a phosphorothioate/phosphodiesterderivative at 300 μM for 6 hours and then infected with HIV-1 at amultiplicity of infection (M.O.I) of approximately 0.1. Adenosine andadenine were tested at 900 μM. At 96 hours post-infection, culturesupernatant was removed and HIV-1 RT activity was assayed in triplicateas described by Henderson et al., Virology 182:186-198 (1991). Brieflyin this method, 25 μl of culture supernatant was added to a 50 μlcocktail containing 50 mM Tris (pH 8.0), 20 mM dithiothreitol, 10 mMMgCl₂, 60 mM NaCl, 0.05 Nonidet p-40, 5 μg/ml oligodeoxythymidylic acid,10 μg/ml polyriboadenylic acid, 10 μM deoxythymidine triphosphate and 1mCi ³² p!thymidine 5'-triphosphate. The mixture was incubated at 37° C.for 2 hours. Fifty microliters of the cocktail were then spotted ontodiethylaminoethyl (DEAE) paper, dried, washed with 2× SSC solution(three times for 10 minutes each time) and 95% ethanol (two times for 5minutes each time), dried and exposed to radiographic film for 18 to 24hours at -80° C. The filters were cut and final quantitation wasdetermined by scintillation spectrometry.

The data for the HIV-1 RT activity is shown in Table 2. As indicated,the trimer ApA_(Sp) *A was the most efficient inhibitor of HIV-1 RTactivity (78%). On the contrary, its PR enantiomer, ApA_(Rp) *Ainhibited HIV-1 reverse transcription by 31%. Similarly, the tetramerwith the PS phosphorothioate/phosphodiester linkage adjacent to the 2'terminal linkage, ApApA_(Sp) *A, was able to suppress RT activity 62%while its PR counterpart was only 38% effective in inhibiting thisactivity.

                  TABLE 2    ______________________________________    Inhibition of HIV-1 Reverse Transcriptase Activity    by Phosphorothioate/Phosphodiester 2-5A    2-5A or       Percent Inhibition of HIV-1    Derivative    Reverse Transcriptase.sup.1    ______________________________________    2',5'-A.sub.Rp *ApA                  32    2',5'-A.sub.Sp *ApA                  56    2',5'-ApA.sub.Rp *A                  31    2',5'-ApA.sub.Sp *A                  78    2',5'-A.sub.Rp *ApApA                  57    2',5'-A.sub.Sp *ApApA                  54    2',5'-ApA.sub.Rp *ApA                  52    2',5'-ApA.sub.Sp *ApA                  42    2',5'-ApApA.sub.Rp *A                  38    2',5'-ApApA.sub.Sp *A                  62    2',5'-A.sub.4 26    A.sub.4        8    Adenosine      0    Adenine        4    ______________________________________     .sup.1 Average of triplicate determinations. Intraassay variation for     replicates was <10%.

The compounds of the present invention may be combined with appropriatepharmaceutical or agricultural carriers to form an antiviralcomposition.

For pharmaceutical use, the compounds of the invention may be taken upin pharmaceutically acceptable carriers, such as, solutions,suspensions, tablets, capsules, ointments, elixirs and injectablecomposition and the like. They are administered to subjects sufferingfrom viral infection. The dosage administered depends upon the natureand severity of the infection, the disease stage, and, when administeredsystematically, the size and weight of the infected subject.

The compounds are generally administered in the form of water-solublesalts. Pharmaceutically acceptable water soluble salts include, forexample, the sodium, potassium or ammonium salts of the activecompounds. They are readily dissolved in water or saline solution. Thus,the preferred formulation for pharmacological use comprises a salinesolution of the desired compound in salt form. The formulation mayfurther contain an agent, such as a sugar or protein, to maintainosmotic balance. The salt form of the compound is preferred owing to therelatively high acidity (about pH 3) of the acid form of the compounds.

The compounds of the invention may be used as a treatment orprophylactically for humans and animals from viral infectives such asHerpes simplex, rhinovirus, hepatitis and other infections of thehepatitis virus family, Epstein Barr virus, measles virus, multiplesclerosis (which may be caused by a viral agent) and the various HumanT-Lymphotropic Viruses ("HTLV"), such as HTLV-1, which causes cutaneousT cell lymphoma, HTLV-2, which causes Sezary lymphoma, and HTLV-3, whichis responsible for Acquired Immune Deficiency Syndrome ("AIDS"). Thecompounds of the invention inhibit the HIV-1 Induced Syncytia formation.

The compounds may be applied topically to treat skin cancers caused byradiation, carcinogens or viral agents. Such skin cancers includecutaneous T-cell lymphoma, Sezary lymphoma, Xeroderma pigmentosium,ataxia telangiectasia and Bloom's syndrome. A sufficient amount of apreparation containing a compound of the invention is applied to coverthe lesion or affected area. An effective concentration of active agentis between about 10⁻³ M and 10⁻⁵ M, with 10⁻⁴ M being preferred.

The compounds of the present invention may also be used to treatplant-infecting virus, particularly tobacco mosaic virus, and otherviruses which cause necrosis in turnips, cucumber, orchids and in otherplants. Such viruses include, but are not limited to, tobacco veinmottling virus, vesicular stomatitis virus, vaccinia virus, turnipnecrosis virus, and cymbidium orchid virus.

The compounds may be administered effectively to plants by topicalapplication by abrasion of the leaf surface, aerosol spray, treatment ofthe soil, spraying, or dusting.

An effective antiviral composition may be formed by combining one ormore of the compounds of the invention with a carrier material suitablefor agricultural use. While the individual stereoisomers are preferredfor pharmaceutical use, mixtures of one or more of stereoisomers may beemployed in agricultural applications. The active compound may also beadministered by spraying insect vectors such as aphids, thrips andwhiteflies which carry virus to plants. The dosage administered dependsupon the severity of the infection.

The compounds of the invention may be applied to plant seeds prior togermination to control viruses contained in the germ plasm. The seedsmay be soaked in a solution of polyethylene glycol ("PEG") containingone or more of the compounds. PEG brings the seeds to physiologicalactivity and arrest. The relative concentration of active compound toPEG depends upon the type of seed under treatment.

Plants may be effectively treated with an aqueous formulation containingfrom about 10⁻¹ to about 10⁻² M concentration of active ingredient. Thecompounds of the invention may be applied at very low concentrations. Aneffective amount of active ingredient on the plant surface is from about10⁻⁸ to about 10⁻¹² mole per cm² of being preferred. For the typicaltobacco plant of 1,000 cm², 10⁻⁵ M of compound is effective. At thisrate, one pound of active ingredient is sufficient to treat 2×10⁸tobacco plants.

For agricultural application, the compounds are advantageouslyadministered in the form of water-soluble salts, e.g. ammonium orpotassium salts. Sodium salts are generally avoided in treating edibleplants.

The compounds of the invention are readily dissolved in water,particularly at such low concentrations. Aqueous formulations foragricultural use may optionally contain a sticker and/or aUV-stabilizer. Such agents are well-known to those skilled in the art.Fatty acids (1%) are useful as spreader sticker agents. EffectiveUV-stabilizers include, for example, p-aminobenzoic acid.

For antiviral use in mammals, the compounds of the invention areadministered parenterally, such as intravenously, intraarterially,intramuscularly, subcutaneously or when administered as an anti-canceragent, intratumorally. The preferred route of administration forantiviral therapy is intravenous injection. The compounds of theinvention may be administered to mammals at very low concentrations. Theactual dosage administered may take into account the size and weight ofthe patient, whether the nature of the treatment is prophylactic ortherapeutic in nature, the age, health and sex of the patient, the routeof administration, the nature and stage of the affliction, and otherfactors. An effective daily dosage of active ingredient, based upon invivo studies involving other 2-5A analogues, is from about 0.25 g per 70kg of body weight (approximately 152 lbs) to about 2.5 g per 70 kg ofbody weight. The preferred daily dosage is about 0.5 g per 70 kg of bodyweight. Those skilled in the art should readily be able to deriveappropriate dosages and schedules of aministration to suit the specificcircumstance and needs of the patient.

It is expected that an effective treatment regimen includesadministration of the daily dosage for two days. Treatment is continuedat least until the disease condition is substantially abated.

Preferably, the therapeutic end point is determined by testing for thecontinued presence of viral DNA. Such testing can be done by polymerasechain reaction (PCR) in which the presence of viral DNA is assayedaccording to convential PCR. PCR primers of appropriate nucleotidesequences for amplification of viral DNA can be prepared from knownviral nucleotide sequences. To obtain DNA for testing, patientperipheral blood mononuclear cells are lysed with an appropriate lysingagent, such as NP-40.

Alternatively, testing for the continued presence of the virus can beperformed by an antigen-antibody assay using any of the known monoclonalor polyclonal antisera against a protein antigen of the target virus'protein coat. For example, an antigen-antibody assay may be employed todetect any of the protein antigen in the antigens HIV protein coat, forexample, the gp120, p17 or p24. Moreover, the target antigen is notlimited merely to coat protein antigens. Antisera can be targetedagainst a suitable non-coat protein antigen, such as the HIV reversetranscriptase (RT) molecule. Monoclonal antibodies to HIV RT are known.Sobol et al., Biochemistry 30: 10623-10631 (1991).

Additionally, testing for the presence of the infecting virus during orpost-treatment could be accomplished by an assay which assesses theviral load in the patient's blood stream. This can be done bydetermining the level of syncytia formation, i.e., by measuring theformation of viral particles. See procedure outlined in Henderson etal., Virology 182: 186-198 (1994).

In addition to administration with conventional carriers, the compoundsof the present invention may be administered by a variety of specializedoligonucleotide or nucleic acid delivery techniques. 2-5A and itsanalogues have been successfully encapsulated in various encapsulatingmaterials, such as in unilamellar liposomes and delivered with the aidof monoclonal antibodies to cells, Bayard et al., Eur. J. Biochem.151:319-325 (1985). Reconstituted Sendai virus envelopes have beensuccessfully used to deliver RNA and DNA to cells, Arad et al., Biochem.Biophys. Acta. 859: 88-94 (1986). Moreover, the virus envelope is notlimited to Sendai virus, but could include encapsulation in anyretroviral amphotrophic particle. For example, an HIV envelope could beformed from any part or all of the outer protein coat of anon-infectious HIV particle. Such particles as gp 120 can be cloned byknown recombinant techniques. These techniques may be utilized forintroduction of the present 2',5'-phosphorothioate/phosphodiesteroligoadenylates into cells. It is further contemplated that thecompounds of the invention may be administered in the form of prodrugsin which lipophilic groups are attached to, for example, the 5'-terminalhydroxyl group of the core compound.

Conjugation of 2',5'-Phosphorothioate Tetramer Adenylates

The 2',5'-phosphorothioate/phosphodiester tetramers of the invention maybe conjugated with the carrier (poly)L-lysine. (Poly)L-lysine has beenshown to be an effective vector for introducing 2',5'-oligoadenylatesand analogues into intact cells. Bayard et al., Biochemistry 25:3730-3736 (1986) Poly(L-lysine) conjugation to trimer molecules is notfeasible, owing to the destruction of the 2 -terminal ribosyl moiety andsubsequent inactivation of the molecule. Conjugation to poly(L-lysine)permits efficient intracellular transport of the2',5'-phosphorothioate/phosphodiester oligoadenylates of the invention,while preserving intact within the conjugate the trimer moiety believednecessary for good biological activity.

The conjugates are formed by introducing two aldehyde functions at the2' end of the tetramer by periodate oxidation of the alpha-glycol groupof the ribose residue. The resulting aldehyde groups are then randomlycoupled to the epsilon-amino groups of lysine residues of poly(L-lysine)by Schiff base formation, and then reduced with sodium cyanoborohydrideat pH 8.0. This procedure converts the 2',3'-terminal ribose ring into amorpholine structure. The poly(L-lysine) peptide preferably containsfrom about 60 to about 70 lysine residues. From about five to about tenof the lysine residues are coupled in this manner to tetramer moieties.The resulting 2',5'-phosphorothioate/phosphodiester/(poly)L-lysineconjugates may then be isolated by gel filtration chromatography on aSephadex G-50 column.

The 2',5'-phosphorothioate/phosphodiester oligoadenylate poly(L-lysine)conjugates have the formula: ##STR12## wherein q is an integer fromabout 60 to about 70 and each R is independently R' or ##STR13## Fromabout five to about ten of the R groups comprise R'. The R' group hasthe following formula: ##STR14## wherein m is zero, 1, 2 or 3; and R₃,R₄ and R₅ are independently selected from the group of oxygen andsulfur, provided that all R₃, R₄ and R₅ may not be oxygen, and furtherprovided that all R₃, R₄ and R₅ may not be sulfur.

The conjugates may be advantageously prepared by the procedure of Bayardet al., Biochemistry 25: 3730-3736 (1986):

EXAMPLE 7 Preparation ofPoly(L-lysine)/2',5'-Phosphorothioate/Phosphodiester OligoadenylateConjugates

A 4-microliter aliquot of sodium metaperiodate (0.6 micromole in 0.1Msodium acetate buffer, pH 4.75) is added to an ice-cold solution of2',5'-phosphorothioate/phosphodiester tetramer adenylate in 400microliter of distiller water. The reaction mixture is stirred on icefor 30 min; 400 microliter of poly(L-lysine) (0.14 micromole in 0.2Mphosphate buffer, pH 8.0) and 200 microliter of sodium cyanoborohydride(20 micromole in 0.2M phosphate buffer, pH 8.0) are added. The mixtureis incubated for 2 h at room temperature and then loaded on a SephadexG-50 column equilibrated with 0.1M sodium acetate buffer, pH 4.75. Eachfraction is assayed for its phosphorothioate/phosphodiesteroligoadenylate/poly(L-lysine) content by the method described by Lowryet al., J. Biol. Chem. 193:265-275 (1951), and by absorbance at 260 nm.

Conjugation of the 2',5'-phosphothioate/phosphodiester tetramer topoly(L-lysine) leaves the remaining three 2',5'-linkedphosphorothioate/phosphodiester adenylic residues intact for optimalRNase L binding and activation.

Liposome Encapsulation of 2',5'Phosphorothioate/phosphodiesterOligoadenylates

Encapsulation of the compounds of the present invention comprisesanother attractive non-disruptive technique for introduction into cells.Liposome encapsulation may be advantageously accomplished according tothe technique described by Kondorosi et al., FEBS Lett. 120:37-40(1980).

EXAMPLE 8 Preparation of Large Unilamellar Vesicles (Liposomes) Loadedwith 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates

Briefly, a phospholipid mixture from bovine brain (Sigma Chemical Co.,Folch fraction III composed of 80-85% phosphatidylserine with theremaining 15% composed of other brain lipids; 35 mg) is suspended in 5ml of buffer A 0.1M NaCl, 2 mM histidine, 2 mMN-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acit ("TES"), 0.4 mMEDTA (pH 7.4) by vortexing. The suspension is sonicated under nitrogenfor 10 minutes at 0° C. The suspension is further incubated for 1 hr at37° C. after adjusting the final concentration of Ca⁺⁺ to 20 mM by theaddition of 125 microliters of 800 mM CaCl₂. The resulting precipitateis sedimented by centrifugation (2500×g, 10 min), vortexing and mixingwith 100 microliters of 1×10⁻⁴ M 2',5'-phosphorothioate/phosphodiesteroligoadenylate, which is dissovled in phosphate-buffered saline. Thefinal concentration of EDTA is then adjusted to 120 mM by the additionof 400 microliters of buffer B 150 mM EDTA, pH 7.4, 0.1M NaCl, 2 mMhistidine, 2 mM TES!. Liposomes are formed after incubation of thismixture for 30 minutes at 37° C. The excess of EDTA and non-encapsulatedcomponents are removed by passing the liposomes through a Sephadex G-25column which is equilibrated with phosphate-buffered saline. About 10%of the 2',5'-phosphorothioate/phosphodiester oligoadenylate isencapsulated into liposomes by this procedure. The liposome suspensionis stable at 4° C. for one week following preparation.

Preparation of Reconstituted Sendai Virus Envelopes Containing2',5'-Phosphorothioate/Phosphodiester Oligoadenylates

Reconstituted Sendai virus envelopes may be used as efficient vehiclesfor the introduction of polynucleotides into cells. Arad et al.,Biochimica et Biophysica Acta 859: 88-94 (1986), discloses introductionof poly(I) poly(C) into cultured cells by the use of reconstitutedSendai virus envelopes. Fusion of the aforesaid reconstituted Sendaivirus envelopes leads to introduction of the enclosed macromoleculesinto the recipient cell cytoplasm. Reconstituted Sendai virus envelopesmay be obtained by detergent solubilization of intact Sendai virusparticles. The reconstituted envelopes are fusogenic vesicles consistingof the viral envelope phospholids and their glycoproteins, devoid of theviral genomic RNA.

Incorporation of the compounds of the present invention intoreconstituted Sendai virus envelopes for fusion-mediated micro-injectionmay be accomplished by following the procedure of Arad et al.,Biochimica et Biophysica Acta 859: 88-94 (1986). Briefly, a pellet ofSendai virus particles (1.5 mg protein) is dissolved in 30 microlitersof a solution containing 10% Triton X-100, 100 mM NaCl, 50 mM Tris-HCl(pH 7.4) and 0.1 mM phenylmethylsulfonyl fluoride (Triton X-100:proteinratio, 2:1, w/w). To the clear supernatant obtained aftercentrifugation, 2',5'-phosphorothioate/phosphodiester oligoadenylatedissolved in a solution A (160 mM NaCl, 20 mM Tris-HCl (pH 7.4)) isadded to give a final concentration of active ingredient of 5-20 mg/mland a final volume of 150 microliters. Triton X-100 is removed from thesupernatant by direct addition of 40 mg of SM-2 Bio-Beads. The turbidsuspension obtained (containing reconstituted Sendai virus envelopes) iscentrifuged at 100,000×g for 1 h. The pellet, containing about 10% ofthe original viral protein, is then suspended in solution A to give afinal protein concentration of 25 micrograms/ml.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

We claim:
 1. A compound of the formula: ##STR15## wherein m is zero, 1,2 or 3; and R, R₁ and R₂ are independently selected from the groupconsisting of oxygen and sulfur, provided that all R, R₁ and R₂, may notbe oxygen, and further provided that all R, R₁ and R₂ may not be sulfur;or water-soluble salt thereof.
 2. A compound according to claim 1wherein m is
 1. 3. A compound according to claim 1 wherein m is
 0. 4. Acompound according to claim 1 selected from the group consisting ofadenylyl-(2',5')-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine, the5'-mono-, di-, and triphosphates thereof, and water-soluble salts of anyof them.
 5. A compound according to claim 1 selected from the groupconsisting ofadenylyl-(2',5')-adenylyl-(2',5')-P-thioadenylyl-(2',5')-adenosine, the5'-mono-, di-, and triphosphates thereof, and water-soluble salts of anyof them.
 6. A compound according to claim 1 selected from the groupconsisting ofadenylyl-(2',5')-P-thioadenylyl-(2',5')-P-thioadenylyl-(2',5')-adenosine,the 5'-mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 7. An antiviral composition comprising a compound accordingto claim 1 in combination with a pharmaceutical or agricultural carrier.8. An antiviral composition according to claim 7 wherein m is
 1. 9. Anantiviral composition according to claim 7 wherein the carrier comprisesan encapsulating material selected from the group consisting ofreconstituted Sendai virus envelope and liposome, which materialencapsulates the compound.
 10. A method of treating viral infection in amammal comprising administering thereto an antiviral effective amount ofa compound according to claim
 1. 11. A method according to claim 10wherein m is
 1. 12. A pharmaceutical composition comprising apharmaceutical carrier and a compound according to claim
 1. 13. Anisolated optical isomer according to claim 1, or water-soluble salt ofsuch isolated isomer.
 14. An isolated optical isomer according to claim13 having one or two internucleotide phosphorothioate groups ##STR16##at least one of which is of the PR configuration.
 15. An isomeraccording to claim 14 selected from the group consisting ofadenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine,the 5' mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 16. An isomer according to claim 13 selected from the groupconsisting ofadenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine,the 5' mono-, di-, triphosphates thereof, and water-soluble salts of anyof them.
 17. An isomer according to claim 14 selected from the groupconsisting ofadenylyl-(2',5')-adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenosine,the 5' mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 18. An isomer according to claim 13 selected from the groupconsisting ofadenylyl-(2',5')-adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine,the 5' mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 19. A method of treating viral infection in a mammalcomprising administering thereto an antiviral effective amount of anisolated optical isomer according to claim
 13. 20. A pharmaceuticalcomposition comprising a pharmaceutical carrier and an isolated opticalisomer according to claim
 13. 21. A conjugate of poly(L-lysine) and a2'-5' phosphorothioate/phosphodiester oligoadenylate, said conjugatehaving the formula ##STR17## wherein q is an integer from about 60 toabout 70 and each R is independently R' or ##STR18## provided from aboutfive to about ten of the R groups are R', which R' has the followingformula ##STR19## wherein m is zero, 1, 2, 3; and where R₃, R₄ and R₅,are independently selected from the group of oxygen and sulfur, providedthat all R₃, R₄ and R₅ may not be oxygen, and further provided that allR₃, R₄ and R₅ may not be sulfur.
 22. A method of treating viralinfection in a plant comprising administering thereto an antiviraleffective amount of a compound according to claim
 1. 23. A method oftreating viral infection in a plant comprising administering thereto anantiviral effective amount of a compound according to claim
 2. 24. Amethod of treating viral infection in a plant comprising administeringthereto an antiviral effective amount of an optical isomer according toclaim
 13. 25. A method according to claim 10 wherein the viral infectiontreated is infection by human immunodeficiency virus.
 26. A methodaccording to claim 19 wherein the viral infection treated is infectionby human immunodeficiency virus.
 27. A compound of the formula:##STR20## wherein m is zero, 1, 2 or 3; or water-soluble salt thereof.28. An isomer according to claim 27 wherein m is
 1. 29. An isomeraccording to claim 27 wherein m is
 0. 30. An isolated optical isomeraccording to claim
 27. 31. An antiviral composition comprising acompound according to claim 27 in combination with a pharmaceutical oragricultural carrier.
 32. A method of treating viral infection in aplant comprising administering thereto an antiviral effective amount ofa compound according to claim
 27. 33. A method of treating viralinfection in a mammal comprising administering thereto an antiviraleffective amount of a compound according to claim
 27. 34. A methodaccording to claim 33 wherein the viral infection treated is infectionby human immunodeficiency virus.
 35. An antiviral composition comprisinga pharmaceutical or agricultural carrier in combination with a compoundof the formula ##STR21## wherein m is zero, 1, 2 or 3; or water-solublesalt thereof.
 36. A composition according to claim 35 wherein m is 1.37. A composition according to claim 35 wherein m is
 0. 38. Acomposition according to claim 35 wherein the compound is an isolatedoptical isomer.
 39. A composition according to claim 38 wherein theoptical isomer is selected from the group consisting ofadenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')adenosine, the 5' mono-,di-, and triphosphates thereof, and water-soluble salts of any of them.40. A composition according to claim 39 wherein the optical isomer isadenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')adenosine, or water-solublesalt thereof.
 41. A composition according to claim 38 wherein theoptical isomer is selected from the group consisting ofadenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')adenosine, the 5' mono-,di-, and triphosphates thereof, and water-soluble salts of any of them.42. A composition according to claim 41 wherein the optical isomer isadenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')adenosine, or water-solublesalt thereof.
 43. A method of treating viral infection in a plantcomprising administering thereto an antiviral effective amount of acomposition according to claim
 35. 44. A method of treating viralinfection in a mammal comprising administering thereto an antiviraleffective amount of a composition according to claim
 35. 45. A method oftreating viral infection in a mammal according to claim 44 wherein thecompound contained in the composition isadenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')adenosine, or water-solublesalt thereof.
 46. A method of treating viral infection in a mammalaccording to claim 44 wherein the compound contained in the compositionis adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')adenosine, orwater-soluble salt thereof.
 47. A method according to claim 44 whereinthe viral infection treated is infection by human immunodeficiencyvirus.
 48. A method of treating viral infection in a mammal comprisingadministering thereto an antiviral effective amount of a compositionaccording to claim 20.